Immunological herpes simplex virus antigens and methods for use thereof

ABSTRACT

The invention provides HSV antigens that are useful for the prevention and treatment of HSV infection. Disclosed herein are antigens and/or their constituent epitopes confirmed to be recognized by T-cells derived from herpetic lesions or from uterine cervix. T-cells having specificity for antigens of the invention have demonstrated cytotoxic activity against cells loaded with virally-encoded peptide epitopes, and in many cases, against cells infected with HSV. The identification of immunogenic antigens responsible for T-cell specificity provides improved anti-viral therapeutic and prophylactic strategies. Compositions containing antigens or polynucleotides encoding antigens of the invention provide effectively targeted vaccines for prevention and treatment of HSV infection.

This application claims the benefit of U.S. provisional patentapplications 60/095,723 and 60/095,724, both filed on Aug. 7, 1998, theentire contents of which are incorporated herein by reference.

The invention disclosed herein was made with Government support underGrant No. AI34616, awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to describemore fully the state of the art to which this invention pertains.

TECHNICAL FIELD OF THE INVENTION

The invention relates to molecules, compositions and methods that can beused for the treatment and prevention of herpes simplex virus (HSV)infection. More particularly, the invention identifies epitopes of HSVproteins that can be used for the development of methods, molecules andcompositions that stimulate or augment HSV-specific immunity.

BACKGROUND OF THE INVENTION

The complete, known DNA sequence of HSV types 1 and 2 are approximately160 kb and encodes about 85 genes, each of which encodes at least oneprotein. Unknown are the immunological epitopes within these proteins,each epitope approximately 9-12 amino acids in length, that are capableof eliciting an effective T cell immune response to viral infection.Cellular immune responses are required to limit the severity ofrecurrent HSV infection in humans. HSV-specific CD4 T cells can becytotoxic towards virally-infected cells (M. Yasukawa et a., 1991, J.Immunol., 146:1341-1347; M. Yasukawa et al., 1984, J. Immunol.,133:2736-42). HSV-specific T cells can also reduce the titer of HSVreplication in HSV-infected, HLA-matched cells, produce lymphokines withantiviral or immunomodulatory activity, or provide specific B cell helpto augment antiviral antibody responses. References relating to theantigenic specificity of HSV-specific T cells include: A. G. Langenberget al., 1995, Ann. Int. Med. 122:889-898; A. Mikloska et al., 1998, J.Gen. Virol., 79:353-361; J. W. Torseth et al., 1987, J. Virol.,61:1532-1539; M. Yasukawa et al., 1985, J. Immunol., 134:2679-2687.

There remains a need to identify specific epitopes capable of elicitingan effective immune response to HSV infection. Such information can leadto the identification of more effective immunogenic antigens useful forthe prevention and treatment of HSV infection.

SUMMARY OF THE INVENTION

The invention provides HSV antigens, polypeptides comprising HSVantigens, polynucleotides encoding the polypeptides, vectors, andrecombinant viruses containing the polynucleotides, antigen-presentingcells (APCs) presenting the polypeptides, immune cells directed againstHSV, and pharmaceutical compositions. The pharmaceutical compositionscan be used both prophylactically and therapeutically. The antigens ofthe invention are recognized by T cells recovered from herpetic lesions.The invention additionally provides methods, including methods forpreventing and treating HSV infection, for killing HSV-infected cells,for inhibiting viral replication, for enhancing secretion of antiviraland/or immunomodulatory lymphokines, and for enhancing production ofHSV-specific antibody. For preventing and treating HSV infection, forenhancing secretion of antiviral and/or immunomodulatory lymphokines,for enhancing production of HSV-specific antibody, and generally forstimulating and/or augmenting HSV-specific immunity, the methodcomprises administering to a subject a polypeptide, polynucleotide,recombinant virus, APC, immune cell or composition of the invention. Themethods for killing HSV-infected cells and for inhibiting viralreplication comprise contacting an HSV-infected cell with an immune cellof the invention. The immune cell of the invention is one that has beenstimulated by an antigen of the invention or by an APC that presents anantigen of the invention. A method for producing such immune cells isalso provided by the invention. The method comprises contacting animmune cell with an APC, preferably a dendritic cell, that has beenmodified to present an antigen of the invention. In a preferredembodiment, the immune cell is a T cell such as a CD4+ or CD8+ T cell.

In one embodiment, the invention provides a composition comprising anHSV polypeptide. The polypeptide can comprise a U_(L)19, U_(L)21,U_(L)49 or U_(L)50 protein or a fragment thereof, or a polypeptideselected from the group consisting of: amino acids 1078-1319 of U_(L)19;amino acids 148-181 of U_(L)21; amino acids 105-190 or 177-220 ofU_(L)49; amino acids 118-312 of U acids 1-273 of glycoprotein E (gE);amino acids 185-197, 209-221 or 430-449 of VP16; and substitutionalvariants of the above. Also provided is an isolated polynucleotide thatencodes a polypeptide of the invention, and a composition comprising thepolynucleotide. The invention additionally provides a recombinant virusgenetically modified to express a polynucleotide of the invention, and acomposition comprising the recombinant virus. In preferred embodiments,the virus is a vaccinia virus, canary pox virus, HSV, lentivirus,retrovirus or adenovirus. A composition of the invention can be apharmaceutical composition. The composition can optionally comprise apharmaceutically acceptable carrier and/or an adjuvant.

The invention additionally provides a method of identifying animmunogenic epitope of an infectious organism, such as a virus, bacteriaor parasite. In one embodiment, the method comprises preparing acollection of random fragments of the organismal genome. The methodfurther comprises expressing a polypeptide encoded by a fragment of thecollection, and recovering the expressed polypeptide. Preferably, thepolypeptide is expressed as an insoluble inclusion body. In oneembodiment, the polypeptide is expressed as a fusion protein using, forexample, a pUEX vector to express an insoluble β-galactosidase fusionprotein. The ability of the expressed polypeptide to elicit a cellularimmune response is then assayed. Ability to elicit a cellular immuneresponse is indicative of the presence of an immunogenic epitope.

The above steps can be repeated with subfragments of the genomefragments. The method can further comprise sequencing a fragment of thegenome. In one embodiment, the assaying comprises performing a T cellproliferation assay. The assaying can be performed with an immune cellderived from a subject that has been exposed to the infectious organism.In preferred embodiments, the cell is derived from a site of activeinfection, such as skin or cervix, or from blood of an infected subject.

The invention further provides immunogenic epitopes identified by themethod of the invention, polypeptides comprising the epitopes, andpolynucleotides encoding the polypeptides. Suitable infectious organismsinclude bacteria, parasites and viruses. Examples of viruses include DNAand RNA viruses, both double-stranded and single-stranded. The method ofthe invention provides a strategy for combating a variety of infectiousorganisms, including those which exhibit significant variability, asknowledge of the organism's nucleic acid sequence is not required.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic representing organization of the HSV genome inthe region of 0.67-0.73 map units. Boundaries are approximate.HSV-1×HSV-2 intertypic recombinant viruses (IRV) are also shown. HSV-2DNA is indicated by a solid line; HSV-1 DNA by a dashed line, andindeterminate regions by a multiple line. The HSV-2 BamH I w fragmentused for expression cloning is also shown.

FIG. 1B is a bar graph showing proliferative responses of T-cell clones(TCC) to the indicated IRV. Data are delta CPM [³H] thymidineincorporation compared to media alone, which was less than 500 cpm ineach case.

FIG. 2 is an immunoblot showing determination of the HSV viral phenotypeof the U_(L)49 gene product (VP22) of IRV DX32. Lysates of mock-infectedcells and cells infected with the viral strains DX32, HSV-1 or HSV-2were separated by SDS-PAGE, blotted, and probed with VP22-specific mAb.The molecular weights kD) of marker proteins are shown at right.

FIG. 3A is a bar graph showing T-cell proliferation elicited by variouspeptide epitopes in VP22 of HSV-2 using TCC 4.2E1. Antigen-presentingcells (APC) were autologous EBV-LCL. Antigens included β-galactosidaseand fusion proteins used at 10 μg/ml and peptides used at 3 μM. Data aredelta cpm [³H] thymidine incorporation compared to media alone, whichwas less than 500 cpm in each case.

FIG. 3B is a bar graph showing T-cell proliferation elicited by variouspeptide epitopes in VP22 of HSV-2 using TCC 1.L3D5.10.8. APC wereautologous PBMC. Antigens included β-galactosidase and fusion proteinsused at 10 μg/ml and peptides used at 1 μM. Data are delta cpm [³H]thymidine incorporation compared to media alone, which was less than 500cpm in each case.

FIG. 3C is a bar graph showing T-cell proliferation elicited by variouspeptide epitopes in VP22 of HSV-2 using TCC ESL4.9. APC were autologousPBMC. Antigens included β-galactosidase and fusion proteins used at 10μg/ml and peptides used at 1 μM. Data are delta cpm [³H] thymidineincorporation compared to media alone, which was less than 500 cpm ineach case.

FIG. 4 is a line graph showing HLA restriction element for T-cell cloneBM.17 response to peptide 437-449 of VP16 of HSV-2. Proliferativeresponses are plotted versus concentration of viral peptide. Antigenpresenting cells are EBV-LCL that are either autologous (closedcircles), homozygous for HLA DQB1*0501 (open triangles), or homozygousfor HLA DQB1*0201 (squares).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides HSV antigens that are useful for the preventionand treatment of HSV infection. Disclosed herein are antigens and/ortheir constituent epitopes confirmed to be recognized by T-cells derivedfrom herpetic lesions. In some embodiments, T-cells having specificityfor antigens of the invention have demonstrated cytotoxic activityagainst virally-infected cells. The identification of immunogenicantigens responsible for T-cell specificity facilitates the developmentof improved anti-viral therapeutic and prophylactic strategies.Compositions containing antigens or polynucleotides encoding antigens ofthe invention provide effectively targeted vaccines for prevention andtreatment of HSV infection.

Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein, “polypeptide” includes proteins, fragments of proteins,and peptides, whether isolated from natural sources, produced byrecombinant techniques or chemically synthesized. Polypeptides of theinvention typically comprise at least about 8 amino acids.

As used herein, “HSV polypeptide” includes HSV-1 and HSV-2, unlessotherwise indicated. References to amino acids of HSV proteins orpolypeptides are based on the genomic sequence information regardingHSV-2 as described in A. Dolan et al., 1998, J. Virol. 72(3):2010-2021.

As used herein, “substitutional variant” refers to a molecule having oneor more amino acid substitutions or deletions in the indicated aminoacid sequence, yet retaining the ability to be recognized by an immunecell. One method for determining whether a molecule can be recognized byan immune cell is the proliferation assay described in D. M. Koelle etal., 1994, J. Virol. 68(5):2803-2810.

As used herein, “vector” means a construct, which is capable ofdelivering, and preferably expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

The term “nucleic acid” or “polynucleotide” refers to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogs of natural nucleotides that hybridize to nucleic acids in amanner similar to naturally-occurring nucleotides.

As used herein, “antigen-presenting cell” or “APC” means a cell capableof handling and presenting antigen to a lymphocyte. Examples of APCsinclude, but are not limited to, macrophages, Langerhans-dendriticcells, follicular dendritic cells, B cells, monocytes, fibroblasts andfibrocytes. Dendritic cells are a preferred type of antigen presentingcell. Dendritic cells are found in many non-lymphoid tissues but canmigrate via the afferent lymph or the blood stream to the T-dependentareas of lymphoid organs. In non-lymphoid organs, dendritic cellsinclude Langerhans cells and interstitial dendritic cells. In the lymphand blood, they include afferent lymph veiled cells and blood dendriticcells, respectively. In lymphoid organs, they include lymphoid dendriticcells and interdigitating cells. As used herein, each of these celltypes and each of their progenitors is referred to as a “dendriticcell,” unless otherwise specified.

As used herein, “modified” to present an epitope refers toantigen-presenting cells (APCs) that have been manipulated to present anepitope by natural or recombinant methods. For example, the APCs can bemodified by exposure to the isolated antigen, alone or as part of amixture, peptide loading, or by genetically modifying the APC to expressa polypeptide that includes one or more epitopes.

As used herein, “pharmaceutically acceptable salt” refers to a salt thatretains the desired biological activity of the parent compound and doesnot impart any undesired toxicological effects. Examples of such saltsinclude, but are not limited to, (a) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; and saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, furmaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids,naphthalenedisulfonic acids, polygalacturonic acid; (b) salts withpolyvalent metal cations such as zinc, calcium, bismuth, barium,magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or(c) salts formed with an organic cation formed fromN,N′-dibenzylethylenediamine or ethylenediamine; or (d) combinations of(a) and (b) or (c), e.g., a zinc tannate salt; and the like. Thepreferred acid addition salts are the trifluoroacetate salt and theacetate salt.

As used herein, “pharmaceutically acceptable carrier” includes anymaterial which, when combined with an active ingredient, allows theingredient to retain biological activity and is non-reactive with thesubject's immune system. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Preferred diluents for aerosol orparenteral administration are phosphate buffered saline or normal (0.9%)saline.

Compositions comprising such carriers are formulated by well knownconventional methods (see, for example, Remington's PharmaceuticalSciences, Chapter 43, 14th Ed., Mack Publishing Co, Easton Pa. 18042,USA).

As used herein, “adjuvant” includes those adjuvants commonly used in theart to facilitate the stimulation of an immune response. Examples ofadjuvants include, but are not limited to, helper peptide; aluminumsalts such as aluminum hydroxide gel (alum) or aluminum phosphate;Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories,Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway,N.J.); AS-2 (Smith-Kline Beecham); QS-21 (Aquilla); MPL or 3d-MPL (RibiImmunoChem Research, Inc., Hamilton, Mont.); LEIF; salts of calcium,iron or zinc; an insoluble suspension of acylated tyrosine; acylatedsugars; cationically or anionically derivatized polysaccharides;polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A andquil A; muramyl tripeptide phosphatidyl ethanolamine or animmunostimulating complex, including cytokines (e.g., GM-CSF orinterleukin-2, -7 or -12) and immunostimulatory DNA sequences. In someembodiments, such as with the use of a polynucleotide vaccine, anadjuvant such as a helper peptide or cytokine can be provided via apolynucleotide encoding the adjuvant.

As used herein, “a” or “an” means at least one, unless clearly indicatedotherwise.

HSV Polypeptides

In one embodiment, the invention provides an isolated herpes simplexvirus (HSV) polypeptide, wherein the polypeptide comprises a U_(L)19(major capsid antigen, VP5), U_(L)21, U_(L)49 (VP22) or U_(L)50 proteinor a fragment thereof In another embodiment, the invention provides anisolated HSV polypeptide selected from the group consisting of: aminoacids 1078-1319 of U_(L)19; amino acids 148-181 of U_(L)21; amino acids105-190 or 177-220 of U_(L)49; amino acids 118-312 of U_(L)50; aminoacids 1-273 of glycoprotein E (gE; US8); amino acids 185-197, 209-221 or430-449 of VP16; and substitutional variants of the above polypeptides.The references to amino acid residues are made with respect to theproteins of the HSV-2 genome as described in A. Dolan et al., 1998, J.Virol. 72(3):2010-2021.

The polypeptide can be a fusion protein. In one embodiment, the fusionprotein is soluble. A soluble fusion protein of the invention can besuitable for injection into a subject and for eliciting an immuneresponse. Within certain embodiments, a polypeptide can be a fusionprotein that comprises multiple polypeptides as described herein, orthat comprises at least one polypeptide as described herein and anunrelated sequence. A fusion partner may, for example, assist inproviding T helper epitopes (an immunological fusion partner),preferably T helper epitopes recognized by humans, or may assist inexpressing the protein (an expression enhancer) at higher yields thanthe native recombinant protein. Certain preferred fusion partners areboth immunological and expression enhancing fusion partners. Otherfusion partners may be selected so as to increase the solubility of theprotein or to enable the protein to be targeted to desired intracellularcompartments. Still further fusion partners include affinity tags, whichfacilitate purification of the protein.

Fusion proteins may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion protein isexpressed as a recombinant protein, allowing the production of increasedlevels, relative to a non-fused protein, in an expression system.Briefly, DNA sequences encoding the polypeptide components may beassembled separately, and ligated into an appropriate expression vector.The 3′ end of the DNA sequence encoding one polypeptide component isligated, with or without a peptide linker, to the 5′ end of a DNAsequence encoding the second polypeptide component so that the readingframes of the sequences are in phase. This permits translation into asingle fusion protein that retains the biological activity of bothcomponent polypeptides.

A peptide linker sequence may be employed to separate the first and thesecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion protein usingstandard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., 1985, Gene 40:39-46; Murphy et al., 1986, Proc. Natl. Acad. Sci.USA 83:8258-8262; U.S. Pat. Nos. 4,935,233 and 4,751,180. The linkersequence may generally be from 1 to about 50 amino acids in length.Linker sequences are not required when the first and second polypeptideshave non-essential N-terminal amino acid regions that can be used toseparate the functional domains and prevent steric interference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located 5′ to the DNAsequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals arepresent 3′ to the DNA sequence encoding the second polypeptide.

Fusion proteins are also provided that comprise a polypeptide of thepresent invention together with an unrelated immunogenic protein.Preferably the immunogenic protein is capable of eliciting a recallresponse. Examples of such proteins include tetanus, tuberculosis andhepatitis proteins (see, for example, Stoute et al., 1997, New Engl. J.Med., 336:869).

Within preferred embodiments, an immunological fusion partner is derivedfrom protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292, 1986). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionprotein. A repeat portion is found in the C-terminal region starting atresidue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

In some embodiments, it may be desirable to couple a therapeutic agentand a polypeptide of the invention, or to couple more than onepolypeptide of the invention. For example, more than one agent orpolypeptide may be coupled directly to a first polypeptide of theinvention, or linkers that provide multiple sites for attachment can beused. Alternatively, a carrier can be used. Some molecules areparticularly suitable for intercellular trafficking and proteindelivery, including, but not limited to, VP22 Elliott and O'Hare, 1997,Cell 88:223-233; see also Kim et al., 1997, J. Immunol. 159:1666-1668;Rojas et al., 1998, Nature Biotechnology 16:370; Kato et al., 1998, FEBSLett. 427(2):203-208; Vives et al., 1997, J. Biol. Chem.272(25):16010-7; Nagahara et al., 1998, Nature Med. 4(12):1449-1452).

A carrier may bear the agents or polypeptides in a variety of ways,including covalent bonding either directly or via a linker group.Suitable carriers include proteins such as albumins (e.g., U.S. Pat. No.4,507,234, to Kato et al.), peptides and polysaccharides such asaminodextran (e.g., U.S. Pat. No. 4,699,784, to Shih et al.). A carriermay also bear an agent by noncovalent bonding or by encapsulation, suchas within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and4,873,088).

In general, polypeptides (including fusion proteins) and polynucleotidesas described herein are isolated. An “isolated” polypeptide orpolynucleotide is one that is removed from its original environment. Forexample, a naturally-occurring protein is isolated if it is separatedfrom some or all of the coexisting materials in the natural system.Preferably, such polypeptides are at least about 90% pure, morepreferably at least about 95% pure and most preferably at least about99% pure. A polynucleotide is considered to be isolated if, for example,it is cloned into a vector that is not part of the natural environment.

The polypeptide can be isolated from its naturally-occurring form,produced by recombinant means or synthesized chemically. Recombinantpolypeptides encoded by DNA sequences described herein can be readilyprepared from the DNA sequences using any of a variety of expressionvectors known to those of ordinary skill in the art. Expression may beachieved in any appropriate host cell that has been transformed ortransfected with an expression vector containing a DNA molecule thatencodes a recombinant polypeptide. Suitable host cells includeprokaryotes, yeast and higher eukaryotic cells. Preferably the hostcells employed are E. coli, yeast or a mammalian cell line such as COSor CHO. Supernatants from the soluble host/vector systems which secreterecombinant protein or polypeptide into culture media may be firstconcentrated using a commercially available filter. Followingconcentration, the concentrate may be applied to a suitable purificationmatrix such as an affinity matrix or an ion exchange resin. Finally, oneor more reverse phase HPLC steps can be employed to further purify arecombinant polypeptide.

Fragments and other variants having fewer than about 100 amino acids,and generally fewer than about 50 amino acids, may also be generated bysynthetic means, using techniques well known to those of ordinary skillin the art. For example, such polypeptides may be synthesized using anyof the commercially available solid-phase techniques, such as theMerrifield solid-phase synthesis method, wherein amino acids aresequentially added to a growing amino acid chain Merrifield, 1963, J.Am. Chem. Soc. 85:2146-2149). Equipment for automated synthesis ofpolypeptides is commercially available from suppliers such as PerkinElmer/Applied BioSystems Division (Foster City, Calif.), and may beoperated according to the manufacturer's instructions.

Variants of the polypeptide for use in accordance with the invention canhave one or more amino acid substitutions, deletions, additions and/orinsertions in the amino acid sequence indicated that result in apolypeptide that retains the ability to elicit an immune response to HSVor HSV-infected cells. Such variants may generally be identified bymodifying one of the polypeptide sequences described herein andevaluating the reactivity of the modified polypeptide using a knownassay such as a T cell assay described herein. Polypeptide variantspreferably exhibit at least about 70%, more preferably at least about90%, and most preferably at least about 95% identity to the identifiedpolypeptides. These amino acid substitutions include, but are notnecessarily limited to, amino acid substitutions known in the art as“conservative”.

A “conservative” substitution is one in which an amino acid issubstituted for another amino acid that has similar properties, suchthat one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. Amino acid substitutions may generally be madeon the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity and/or the amphipathic nature of theresidues. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes. In a preferredembodiment, variant polypeptides differ from a native sequence bysubstitution, deletion or addition of five amino acids or fewer.Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theimmunogenicity, secondary structure and hydropathic nature of thepolypeptide.

Polynucleotides, Vectors, Host Cells and Recombinant Viruses

The invention provides polynucleotides that encode one or morepolypeptides of the invention. The polynucleotide can be included in avector. The vector can further comprise an expression control sequenceoperably linked to the polynucleotide of the invention. In someembodiments, the vector includes one or more polynucleotides encodingother molecules of interest. In one embodiment, the polynucleotide ofthe invention and an additional polynucleotide can be linked so as toencode a fusion protein.

Within certain embodiments, polynucleotides may be formulated so topermit entry into a cell of a mammal, and expression therein. Suchformulations are particularly useful for therapeutic purposes, asdescribed below. Those of ordinary skill in the art will appreciate thatthere are many ways to achieve expression of a polynucleotide in atarget cell, and any suitable method may be employed. For example, apolynucleotide may be incorporated into a viral vector such as, but notlimited to, adenovirus, adeno-associated virus, retrovirus, or vacciniaor other pox virus (e.g., avian pox virus). Techniques for incorporatingDNA into such vectors are well known to those of ordinary skill in theart. A retroviral vector may additionally transfer or incorporate a genefor a selectable marker (to aid in the identification or selection oftransduced cells) and/or a targeting moiety, such as a gene that encodesa ligand for a receptor on a specific target cell, to render the vectortarget specific. Targeting may also be accomplished using an antibody,by methods known to those of ordinary skill in the art.

The invention also provides a host cell transformed with a vector of theinvention. The transformed host cell can be used in a method ofproducing a polypeptide of the invention. The method comprises culturingthe host cell and recovering the polypeptide so produced. The recoveredpolypeptide can be purified from culture supernatant.

Vectors of the invention can be used to genetically modify a cell,either in vivo, ex vivo or in vitro. Several ways of geneticallymodifying cells are known, including transduction or infection with aviral vector either directly or via a retroviral producer cell, calciumphosphate precipitation, fusion of the recipient cells with bacterialprotoplasts containing the DNA, treatment of the recipient cells withliposomes or microspheres containing the DNA, DEAE dextran,receptor-mediated endocytosis, electroporation, micro-injection, andmany other techniques known to those of skill. See, e.g., Sambrook etal. Molecular Cloning—A Laboratory Manual (2nd ed.) 1-3, 1989; andCurrent Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994Supplement).

Examples of viral vectors include, but are not limited to retroviralvectors based on, e.g., HIV, SIV, murine retroviruses, gibbon apeleukemia virus and other viruses such as adeno-associated viruses (AAVs)and adenoviruses. (Miller et al. 1990, Mol. Cell Biol. 10:4239; J.Kolberg 1992, NIH Res. 4:43, and Cornetta et al. 1991, Hum. Gene Ther.2:215). Widely used retroviral vectors include those based upon murineleukemia virus MuLV), gibbon ape leukemia virus (GaLV), ecotropicretroviruses, simian immunodeficiency virus (SIV), humanimmunodeficiency virus (HIV), and combinations. See, e.g. Buchscher etal. 1992, J. Virol. 66(5):2731-2739; Johann et al. 1992, J. Virol.66(5):1635-1640; Sommerfelt et al. 1990, Virol. 176:58-59; Wilson et al.J. Virol. 63:2374-2378; Miller et al. 1991, J. Virol. 65:2220-2224, andRosenberg and Fauci 1993 in Fundamental Immunology, Third Edition, W. E.Paul (ed.) Raven Press, Ltd., New York and the references therein;Miller et al. 1990, Mol. Cell. Biol. 10:4239; R. Kolberg 1992, J. NIHRes. 4:43; and Cornetta et al. 1991, Hum. Gene Ther. 2:215.

In vitro amplification techniques suitable for amplifying sequences tobe subcloned into an expression vector are known. Examples of such invitro amplification methods, including the polymerase chain reaction(PCR), ligase chain reaction (LCR), Qβ-replicase amplification and otherRNA polymerase mediated techniques (e.g., NASBA), are found in Sambrooket al. 1989, Molecular Cloning—A Laboratory Manual (2nd Ed) 1-3; andU.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods andApplications (Innis et al. eds.) Academic Press Inc. San Diego, Calif.1990. Improved methods of cloning in vitro amplified nucleic acids aredescribed in U.S. Pat. No. 5,426,039.

The invention additionally provides a recombinant microorganismgenetically modified to express a polynucleotide of the invention. Therecombinant microorganism can be useful as a vaccine, and can beprepared using techniques known in the art for the preparation of liveattenuated vaccines. Examples of microorganisms for use as live vaccinesinclude, but are not limited to, viruses and bacteria. In a preferredembodiment, the recombinant microorganism is a virus. Examples ofsuitable viruses include, but are not limited to, vaccinia virus, canarypox virus, retrovirus, lentivirus, HSV and adenovirus.

Compositions

The invention provides compositions that are useful for treating andpreventing HSV infection. The compositions can be used to inhibit viralreplication and to kill virally-infected cells. In one embodiment, thecomposition is a pharmaceutical composition. The composition cancomprise a therapeutically or prophylactically effective amount of apolypeptide, polynucleotide, recombinant virus, APC or immune cell ofthe invention. An effective amount is an amount sufficient to elicit oraugment an immune response, e.g., by activating T cells. One measure ofthe activation of T cells is a proliferation assay, as described in D.M. Koelle et al., 1994, J. Virol. 68(5):2803-2810. In some embodiments,the composition is a vaccine.

The composition can optionally include a carrier, such as apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of pharmaceutical compositions of the present invention.Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, and carriersinclude aqueous isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, preservatives,liposomes, microspheres and emulsions.

The composition of the invention can further comprise one or moreadjuvants. Examples of adjuvants include, but are not limited to, helperpeptide, alum, Freund's, muramyl tripeptide phosphatidyl ethanolamine oran immunostimulating complex, including cytokines. In some embodiments,such as with the use of a polynucleotide vaccine, an adjuvant such as ahelper peptide or cytokine can be provided via a polynucleotide encodingthe adjuvant. Vaccine preparation is generally described in, forexample, M. F. Powell and M. J. Newman, eds., “Vaccine Design (thesubunit and adjuvant approach),” Plenum Press (NY, 1995). Pharmaceuticalcompositions and vaccines within the scope of the present invention mayalso contain other compounds, which may be biologically active orinactive. For example, one or more immunogenic portions of other viralantigens may be present, either incorporated into a fusion polypeptideor as a separate compound, within the composition or vaccine.

A pharmaceutical composition or vaccine may contain DNA encoding one ormore of the polypeptides of the invention, such that the polypeptide isgenerated in situ. As noted above, the DNA may be present within any ofa variety of delivery systems known to those of ordinary skill in theart, including nucleic acid expression systems, bacteria and viralexpression systems. Numerous gene delivery techniques are well known inthe art, such as those described by Rolland, 1998, Crit. Rev. Therap.Drug Carrier Systems 15:143-198, and references cited therein.Appropriate nucleic acid expression systems contain the necessary DNAsequences for expression in the patient (such as a suitable promoter andterminating signal). Bacterial delivery systems involve theadministration of a bacterium (such as Bacillus-Calmette-Guerrin) thatexpresses an immunogenic portion of the polypeptide on its cell surfaceor secretes such an epitope. In a preferred embodiment, the DNA may beintroduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of anon-pathogenic (defective), replication competent virus. Suitablesystems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc.Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. My Acad.Sci. 569:86-103; Flexner et al., 1990, Vaccine 8:17-21; U.S. Pat. Nos.4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91102805; Berkner, 1988,Biotechniques 6:616-627; Rosenfeld et al., 1991, Science 252:431-434;Kolls et al., 1994, Proc. Natl. Acad. Sci. USA 91:215-219; Kass-Eisleret al., 1993, Proc. Natl. Acad. Sci. USA 90:11498-11502; Guzman et al.,1993, Circulation 88:2838-2848; and Guzman et al., 1993, Cir. Res.73:1202-1207. Techniques for incorporating DNA into such expressionsystems are well known to those of ordinary skill in the art. The DNAmay also be “naked,” as described, for example, in Ulmer et al., 1993,Science 259:1745-1749 and reviewed by Cohen, 1993, Science259:1691-1692. The uptake of naked DNA may be increased by coating theDNA onto biodegradable beads, which are efficiently transported into thecells.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administration.Compositions of the present invention may be formulated for anyappropriate manner of administration, including for example, topical,oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268 and 5,075,109.

Such compositions may also comprise buffers (e.g., neutral bufferedsaline or phosphate buffered saline), carbohydrates (e.g., glucose,mannose, sucrose or dextrans), mannitol, proteins, polypeptides or aminoacids such as glycine, antioxidants, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.Alternatively, compositions of the present invention may be formulatedas a lyophilizate. Compounds may also be encapsulated within liposomesusing well known technology.

Any of a variety of adjuvants may be employed in the vaccines of thisinvention. Most adjuvants contain a substance designed to protect theantigen from rapid catabolism, such as aluminum hydroxide or mineraloil, and a stimulator of immune responses, such as lipid A, Bortadellapertussis or Mycobacterium tuberculosis derived proteins. Suitableadjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.);aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine acylated sugars; cationically or anionicallyderivatized polysaccharides; polyphosphazenes biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such as GMCSF or interleukin-2, -7, or -12, may also be used as adjuvants.

Within the vaccines provided herein, the adjuvant composition ispreferably designed to induce an immune response predominantly of theTh1 type. High levels of Th1-type cytokines (e.g., IFN-γ, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6, IL-10 and TNF-β) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, 1989, Ann.Rev. Immunol. 7:145-173.

Preferred adjuvants for use in eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3de-O-acylated monophosphoryl lipid A (3D-MPL), together withan aluminum salt. MPL adjuvants are available from Ribi ImmunoChemResearch Inc. (Hamilton, Mont.) (see U.S. Pat. Nos. 4,436,727;4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (inwhich the CpG dinucleotide is unmethylated) also induce a predominantlyTh1 response. Such oligonucleotides are well known and are described,for example, in WO 96/02555. Another preferred adjuvant is a saponin,preferably QS21, which may be used alone or in combination with otheradjuvants. For example, an enhanced system involves the combination of amonophosphoryl lipid A and saponin derivative, such as the combinationof QS21 and 3D-MPL as described in WO 94/00153, or a less reactogeniccomposition where the QS21 is quenched with cholesterol, as described inWO 96/33739. Other preferred formulations comprises an oil-in-wateremulsion and tocopherol. A particularly potent adjuvant formulationinvolving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion isdescribed in WO 95/17210. Another adjuvant that may be used is AS-2(Smith-Kline Beecham). Any vaccine provided herein may be prepared usingwell known methods that result in a combination of antigen, immuneresponse enhancer and a suitable carrier or excipient.

The compositions described herein may be administered as part of asustained release formulation (i.e. a formulation such as a capsule orsponge that effects a slow release of compound followingadministration). Such formulations may generally be prepared using wellknown technology and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a polypeptide,polynucleotide or antibody dispersed in a carrier matrix and/orcontained within a reservoir surrounded by a rate controlling membrane.Carriers for use within such formulations are biocompatible, and mayalso be biodegradable; preferably the formulation provides a relativelyconstant level of active component release. The amount of activecompound contained within a sustained release formulation depends uponthe site of implantation, the rate and expected duration of release andthe nature of the condition to be treated or prevented.

Any of a variety of delivery vehicles may be employed withinpharmaceutical compositions and vaccines to facilitate production of anantigen-specific immune response that targets HSV-infected cells.Delivery vehicles include antigen presenting cells (APCs), such asdendritic cells, macrophages, B cells, monocytes and other cells thatmay be engineered to be efficient APCs.

Such cells may, but need not, be genetically modified to increase thecapacity for presenting the antigen, to improve activation and/ormaintenance of the T cell response, to have antiviral effects per seand/or to be immunologically compatible with the receiver (i.e., matchedHLA haplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs, including tumor and peritumoral tissues,and may be autologous, allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticimmunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529, 1999). Ingeneral, dendritic cells may be identified based on their typical shape(stellate in situ, with marked cytoplasmic processes (dendrites) visiblein vitro) and based on the a lack of differentiation markers of B cells(CD19 and CD20), T cells (CD3), monocytes (CD14) and natural killercells (CD56), as determined using standard assays. Dendritic cells may,of course, be engineered to express specific cell-surface receptors orligands that are not commonly found on dendritic cells in vivo or exvivo, and such modified dendritic cells are contemplated by the presentinvention. As an alternative to dendritic cells, secreted vesiclesantigen-loaded dendritic cells (called exosomes) may be used within avaccine (Zitvogel et al., 1998, Nature Med. 4:594-600).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce maturation and proliferation of dendriticcells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor, mannose receptor and DEC-205marker. The mature phenotype is typically characterized by a lowerexpression of these markers, but a high expression of cell surfacemolecules responsible for T cell activation such as class I and class IIMHC, adhesion molecules (e.g., CD54 and CD11) and costimulatorymolecules (e.g., CD40, CD80 and CD86). APCs may generally be transfectedwith a polynucleotide encoding a polypeptide (or portion or othervariant thereof such that the polypeptide, or an immunogenic portionthereof, is expressed on the cell surface. Such transfection may takeplace ex vivo, and a composition or vaccine comprising such transfectedcells may then be used for therapeutic purposes, as described herein.Alternatively, a gene delivery vehicle that targets a dendritic or otherantigen presenting cell may be administered to a patient, resulting intransfection that occurs in vivo. In vivo and ex vivo transfection ofdendritic cells, for example, may generally be performed using anymethods known in the art, such as those described in WO 97/24447, or thegene gun approach described by Mahvi et al., 1997, Immunology and CellBiology 75:456-460. Antigen loading of dendritic cells may be achievedby incubating dendritic cells or progenitor cells with the tumorpolypeptide, DNA (naked or within a plasmid vector) or RNA; or withantigen-expressing recombinant bacterium or viruses (e.g., vaccinia,fowlpox, adenovirus or lentivirus vectors). Prior to loading, thepolypeptide may be covalently conjugated to an immunological partnerthat provides T cell help (e.g., a carrier molecule). Alternatively, adendritic cell may be pulsed with a non-conjugated immunologicalpartner, separately or in the presence of the polypeptide.

Administration of the Compositions

Treatment includes prophylaxis and therapy. Prophylaxis or treatment canbe accomplished by a single direct injection at a single time point ormultiple time points. Administration can also be nearly simultaneous tomultiple sites.

Patients or subjects include mammals, such as human, bovine, equine,canine, feline, porcine, and ovine animals.

Compositions are typically administered in vivo via parenteral (e.g.intravenous, subcutaneous, and intramuscular) or other traditionaldirect routes, such as buccal/sublingual, rectal, oral, nasal, topical,(such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial,intraperitoneal, intraocular, or intranasal routes or directly into aspecific tissue.

The compositions are administered in any suitable manner, often withpharmaceutically acceptable carriers. Suitable methods of administeringcells in the context of the present invention to a patient areavailable, and, although more than one route can be used to administer aparticular cell composition, a particular route can often provide a moreimmediate and more effective reaction than another route.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time, or to inhibit infection or diseasedue to infection. Thus, the composition is administered to a patient inan amount sufficient to elicit an effective immune response to thespecific antigens and/or to alleviate, reduce, cure or at leastpartially arrest symptoms and/or complications from the disease orinfection. An amount adequate to accomplish this is defined as a“therapeutically effective dose.”

The dose will be determined by the activity of the composition producedand the condition of the patient, as well as the body weight or surfaceareas of the patient to be treated. The size of the dose also will bedetermined by the existence, nature, and extent of any adverse sideeffects that accompany the administration of a particular composition ina particular patient. In determining the effective amount of thecomposition to be administered in the treatment or prophylaxis ofdiseases such as HSV infection, the physician needs to evaluate theproduction of an immune response against the virus, progression of thedisease, and any treatment-related toxicity.

For example, a vaccine or other composition containing a subunit HSVprotein can include 1-10,000 micrograms of HSV protein per dose. In apreferred embodiment, 10-1000 micrograms of HSV protein is included ineach dose in a more preferred embodiment 10-100 micrograms of HSVprotein dose. Preferably, a dosage is selected such that a single dosewill suffice or, alternatively, several doses are administered over thecourse of several months. For compositions containing HSVpolynucleotides or peptides, similar quantities are administered perdose.

In one embodiment, between 1 and 10 doses may be administered over a 52week period. Preferably, 6 doses are administered, at intervals of 1month, and booster vaccinations may be given periodically thereafter.Alternate protocols may be appropriate for individual patients. Asuitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an antiviral immune response,and is at least 10-50% above the basal (i.e., untreated) level. Suchvaccines should also be capable of causing an immune response that leadsto an improved clinical outcome in vaccinated patients as compared tonon-vaccinated patients. In general, for pharmaceutical compositions andvaccines comprising one or more polypeptides, the amount of eachpolypeptide present in a dose ranges from about 100 μg to 5 mg per kg ofhost. Preferably, the amount ranges from about 10 to about 1000 μg perdose. Suitable volumes for administration will vary with the size, ageand immune status of the patient, but will typically range from about0.1 mL to about 5 mL, with volumes less than about 1 mL being mostcommon.

Compositions comprising immune cells are preferably prepared from immunecells obtained from the subject to whom the composition will beadministered. Alternatively, the immune cells can be prepared from anHLA-compatible donor. The immune cells are obtained from the subject ordonor using conventional techniques known in the art, exposed to APCsmodified to present an epitope of the invention, expanded ex vivo, andadministered to the subject. Protocols for ex vivo therapy are describedin Rosenberg et al., 1990, New England J. Med. 9:570-578. In addition,compositions can comprise APCs modified to present an epitope of theinvention.

Immune cells may generally be obtained in sufficient quantities foradoptive immunotherapy by growth in vitro, as described herein. Cultureconditions for expanding single antigen-specific effector cells toseveral billion in number with retention of antigen recognition in vivoare well known in the art. Such in vitro culture conditions typicallyuse intermittent stimulation with antigen, often in the presence ofcytokines (such as IL-2) and non-dividing feeder cells. As noted above,immunoreactive polypeptides as provided herein may be used to enrich andrapidly expand antigen-specific T cell cultures in order to generate asufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage, monocyte,fibroblast and/or B cells, may be pulsed with immunoreactivepolypeptides or transfected with one or more polynucleotides usingstandard techniques well known in the art. For example,antigen-presenting cells can be transfected with a polynucleotide havinga promoter appropriate for increasing expression in a recombinant virusor other expression system. Cultured effector cells for use in therapymust be able to grow and distribute widely, and to survive long term invivo. Studies have shown that cultured effector cells can be induced togrow in vivo and to survive long term in substantial numbers by repeatedstimulation with antigen supplemented with IL-2 (see, for example,Cheever et al., 1997, Immunological Reviews 157:177).

Administration by many of the routes of administration described hereinor otherwise known in the art may be accomplished simply by directadministration using a needle, catheter or related device, at a singletime point or at multiple time points.

In Vivo Testing of Identified Antigens

Conventional techniques can be used to confirm the in vivo efficacy ofthe identified HSV antigens. For example, one technique makes use of amouse challenge model. Those skilled in the art, however, willappreciate that these methods are routine, and that other models can beused.

Once a compound or composition to be tested has been prepared, the mouseor other subject is immunized with a series of injections. For exampleup to 10 injections can be administered over the course of severalmonths, typically with one to 4 weeks elapsing between doses. Followingthe last injection of the series, the subject is challenged with a doseof virus established to be a uniformly lethal dose. A control groupreceives placebo, while the experimental group is actively vaccinated.Alternatively, a study can be designed using sublethal doses.Optionally, a dose-response study can be included. The end points to bemeasured in this study include death and severe neurological impairment,as evidenced, for example, by spinal cord gait. Survivors can also besacrificed for quantitative viral cultures of key organs includingspinal cord, brain, and the site of injection. The quantity of viruspresent in ground up tissue samples can be measured. Compositions canalso be tested in previously infected animals for reduction inrecurrence to confirm efficacy as a therapeutic vaccine.

Efficacy can be determined by calculating the IC₅₀, which indicates themicrograms of vaccine per kilogram body weight required for protectionof 50% of subjects from death. The IC₅₀ will depend on the challengedose employed. In addition, one can calculate the LD₅₀, indicating howmany infectious units are required to kill one half of the subjectsreceiving a particular dose of vaccine. Determination of the post mortemviral titer provides confirmation that viral replication was limited bythe immune system.

A subsequent stage of testing would be a vaginal inoculation challenge.For acute protection studies, mice can be used. Because they can bestudied for both acute protection and prevention of recurrence, guineapigs provide a more physiologically relevant subject for extrapolationto humans. In this type of challenge, a nonethal dose is administered,the guinea pig subjects develop lesions that heal and recur. Measurescan include both acute disease amelioration and recurrence of lesions.The intervention with vaccine or other composition can be providedbefore or after the inoculation, depending on whether one wishes tostudy prevention versus therapy.

Methods

The invention provides a method for treatment and/or prevention of HSVinfection in a subject. The method comprises administering to thesubject a composition of the invention. The composition can be used as atherapeutic or prophylactic vaccine. In one embodiment, the HSV isHSV-2. Alternatively, the HSV is HSV-1. The invention additionallyprovides a method for inhibiting HSV replication, for killingHSV-infected cells, for increasing secretion of lymphokines havingantiviral and/or immunomodulatory activity, and for enhancing productionof herpes-specific antibodies. The method comprises contacting anHSV-infected cell with an immune cell directed against an antigen of theinvention, for example, as described in the Examples presented herein.The contacting can be performed in vitro or in vivo. In a preferredembodiment, the immune cell is a T cell. T cells include CD4 and CD8 Tcells. Compositions of the invention can also be used as a tolerizingagent against immunopathologic disease, such as eye disease, e.g.,herpes keratitis.

In addition, the invention provides a method of producing immune cellsdirected against HSV. The method comprises contacting an immune cellwith an antigen-presenting cell, wherein the antigen-presenting cell ismodified to present an antigen included in a polypeptide of theinvention. Preferably, the antigen-presenting cell is a dendritic cell.The cell can be modified by, for example, peptide loading or geneticmodification with a nucleic acid sequence encoding the polypeptide. Inone embodiment, the immune cell is a T cell. T cells include CD4 and CD8T cells. Also provided are immune cells produced by the method. Theimmune cells can be used to inhibit HSV replication, to killHSV-infected cells, in vitro or in vivo, to increase secretion oflymphokines having antiviral and/or immunomodulatory activity, toenhance production of herpes-specific antibodies, or in the treatment orprevention of HSV infection in a subject.

The invention provides methods for identifying immunogenic epitopesassociated with infectious organisms. In one embodiment, the methodcomprises preparing a collection of random fragments of the organismalgenome. The preparing can comprise digesting the entire genome, althoughit is not necessary to begin with the full genome. The digestingpreferably comprises contacting the genome with one or more restrictionenzymes to obtain a collection of random fragments having a desiredrange of lengths. Alternatively, one can sonicate, nebulize or otherwisetreat material containing the genome of interest and isolate from a gelfragments of an appropriate size.

The digesting, and the selection of restriction enzymes, is designed toobtain fragments of the genome that are longer than the average lengthof a T cell epitope, e.g., greater than about 30 nucleotides in length.Preferably, the fragments are small enough such that genetic stops areinfrequent, e.g., about 200 to about 500 base pairs in length. Where thegenomic sequence or a restriction map of an organism of interest isknown, one can analyze the genome to identify restriction sites that, iftargeted with the appropriate restriction enzymes, will result in thedesired number of fragments of an appropriate length. The restrictionenzymes can also be selected to target sites that are compatible withsites in a cloning vector to be used.

The random fragments can then be used to express polypeptides encoded bythe fragments. The fragments can be expressed individually, orpreferably, as a pool of polypeptides, either alone or as fusionproteins. Those skilled in the art will appreciate that polypeptides canbe expressed from either DNA or RNA as a starting material. For example,expression of polypeptides from RNA viruses can be achieved by firstpreparing a cDNA from the RNA fragment, and then using the cDNA toexpress the polypeptide. Preferably, the polypeptide is expressed as aninsoluble inclusion body. Expressing the polypeptide as an insolubleinclusion body permits the expression of a large quantity of polypeptidein a form that is readily processed and presented by APCs. Proteinsexpressed as inclusion bodies are easy to purify, provide a highlyefficient method for expression and processing and facilitateapplication of the method to unsequenced organisms.

The polypeptide can be expressed from a vector containing the fragmentof genome. In a preferred embodiment, the vector is a plasmid, such as apUEX vector. Those skilled in the art will appreciate that other vectorscan be used that are capable of expressing polypeptide from an insert.Preferably, the polypeptide is expressed as a fusion protein. Oneexample of a preferred fusion protein is an insoluble β-galactosidasefusion protein. In one embodiment, the expressing comprises culturing ahost cell transformed with a vector containing the fragment of genome.In a preferred embodiment of the method, fragments are ligated intoexpression vectors in the three different reading frames, and in bothdirections.

The method further comprises recovering the expressed polypeptides. Forexample, polypeptide expressed by a cultured host cell can be recoveredby collecting supernatant from the cultured host cell. The recoveredpolypeptide can be further purified from the supernatant using standardtechniques. Polypeptide expressed as an insoluble inclusion body can berecovered by, for example, sonication, lysosyme and detergent-assistedisolation of insoluble inclusion bodies as described in Neophytou etal., 1996, Proc. Natl. Acad. Sci. USA, 93:2014-2018.

The method further comprises assaying the ability of the expressedpolypeptide to elicit an immune response. The ability to elicit animmune response is indicative of the presence of an immunogenic epitopewithin the polypeptide. In one embodiment, the immune response is acellular immune response. The assaying can comprise performing an assaythat measures T cell stimulation or activation. Examples of T cellsinclude CD4 and CD8 T cells.

One example of a T cell stimulation assay is a T cell proliferationassay, such as that described in Example 1 below or in D. M. Koelle etal., 1994, J. Virol. 68(5):2803-2810. The T cell proliferation assay cancomprise, for example, contacting the expressed polypeptide with anantigen-presenting cell and a T cell directed against the virus, andmeasuring T cell proliferation. T cell proliferation can be measured bymeasuring the incorporation of ³H-thymidine or other proliferationmarker. The proliferation assay indicates T cell stimulation ifincreased proliferation is detected in T cells exposed to test antigenas compared to T cell proliferation in response to control antigen. Oneexemplary criterion for increased proliferation is a statisticallysignificant increase in counts per minute (cpm) based on liquidscintillation counting of ³H-thymidine incorporated into precipitatednucleic acid preparations of test as compared to control cell cultures.Another example of assay for T cell stimulation or activation is acytolysis assay. One example of a cytolysis assay is provided in Example1, below.

The assay can be performed on pools of polypeptides to identify poolscontaining active moieties. Further assays can then be performed onincreasingly smaller subsets of the original pools to isolatepolypeptides of interest. The material containing a fragment ofinterest, e.g., a plasmid with its viral insert, can be purified and theviral fragment sequenced. Based on the obtained sequence information,synthetic peptides can be prepared for subsequent testing andconfirmation of the identified antigens. Sequencing of fragments canalso lead to the identification of novel genes.

The foregoing method steps can be repeated, wherein subfragments of thegenome fragments are prepared. Increasingly smaller fragments can beexpressed and tested to determine the minimal epitope.

The method of the invention can be applied to a variety of infectiousorganisms, including bacteria, parasites and viruses. Preferred virusesare those containing intronless DNA or mostly coding sequence. Examplesof viruses include double-stranded DNA viruses, single-stranded DNAviruses, double-stranded RNA viruses and single-stranded RNA viruses.Examples of double-stranded DNA viruses include, but are not limited to,Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex virus-1(HSV-1), HSV-2, varicella-zoster virus (VZV), human herpes virus-6(HHV-6), HHV-7, HHV-8, poxvirus and adenovirus. Examples ofsingle-stranded DNA viruses include, but are not limited to, parvovirus.Examples of double-stranded RNA viruses include, but are not limited to,retroviruses and reoviruses. Examples of single-stranded RNA virusesinclude, but are not limited to, paramyxoviruses, myxoviruses, andflaviviruses.

Because the method does not require knowledge of the organism's nucleicacid sequence, it provides a strategy for combating infectious organismsthat display a great deal of biological variability (e.g., HIV and HCV).For viruses exhibiting high variability, it is advantageous to use asource of viral nucleic acid material derived from a particular patient,a particular site (e.g., blood, skin, cervix) or representative viralstrain circulating in a particular geographical region or patientpopulation, which may differ from prototypical strains of known nucleicacid sequence.

The invention also provides a diagnostic assay. The diagnostic assay canbe used to identify the immunological responsiveness of a patientsuspected of having a herpetic infection and to predict responsivenessof a subject to a particular course of therapy. The assay comprisesexposing T cells of a subject to an antigen of the invention, in thecontext of an appropriate APC, and testing for immunoreactivity by, forexample, measuring IFNγ, proliferation or cytotoxicity. Suitable assaysare described in more detail in the Examples.

EXAMPLES

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Example 1 Identification of Viral Epitopes in HSV-2 Tegument Proteins

This example shows the use of expression cloning with full-length viralDNA to identify T-cell antigens. Described herein are five HSV epitopesrecognized by lesion-infiltrating T-cells discovered by expressioncloning. Also described are several epitopes in VP16 discovered bymethods other than expression cloning.

Viruses and Cells

HSV-1 strain E115 (S. L. Spruance and F. S. Chow, 1980, J. Infect. Dis.,142:671-675.), HSV-2 strain 333 (S. Kit et al., 1983, Biochim. Biophys.Acta., 741:158-170), and intertypic recombinant viruses RS1G31 (M. F.Para et al., 1983, J. Virol., 45:1223-1227), DX32 (V. G. Preston et al.,1978, J. Virol. 28:499-517), and RP-2 (D. M. Koelle et al., 1994, J.Virol., 68:2803-2810) were grown and titered in Vero cells (D. M. Koelleet al., 1993, J. Clin. Invest., 91:961-968). Epstein-Barr virustransformed lymphocyte continuous lines (EBV-LCL) (D. M. Koelle et al.,1993, supra) included autologous lines from donors with genital herpes,AMAI, homozygous for HLA DPB1*0402, HOM2, homozygous for HLA DQB10501,MAT, homozygous for HLA DQB1*0201, and ARENT, homozygous for HLADPB1l2001 (J. G. Bodmer et al., 1996, Tissue Antigens 49:297-321).

HSV-specific T-cells were obtained after approval by the InstitutionalReview Board. Most clones were derived without secondary in vitrostimulation with antigen. Donors 1, 2, and 4 are numbered as previouslydescribed (D. M. Koelle et al., 1994, J. Virol., 68:2803-2810) and werethe sources of lesion-derived clones 1.L3D5.10.8, 2.3 and 4.2E1,respectively; clones 2.3 and 4.2E1 have been previously described (D. M.Koelle et al., 1994, supra). Additional lesion-derived clones came fromdonor ES, from whom clones ESL2.20, ESL3.335, ESL4.34, and ESL4.9 werederived from the second, third, and fourth lesions samples (eachseparated by one year), and donors RH and KM. Clones 2.3, 4.2E1,ESL2.20, RH.13, and KM.17 were derived directly from herpetic vesiclefluid (D. M. Koelle et al., 1994, J. Infect. Dis. 169:956-961). Toderive CD4 TCC ESL4.9, biopsy of a recurrent genital HSV-2 lesion (day 3of symptoms) was performed and bulk lesion-infiltrating cells expandedwith PHA and IL-2 (Schiaperelli Biosystems, Columbia, Md.) in thepresence of acyclovir as described (D. M. Koelle et al., 1998, J. Clin.Invest., 101:1500-09). After 16 days, cells were cloned at 1 cell/well(D. M. Koelle et al., 1994, J. Infect. Dis., 169:956-961). Previouslydescribed VP16-specific clones 1A.B.25, ESL3.334, and ESL4.34 D. G.Doherty et al., 1996, Human Immunol., 47:149; K. R. Jerome et al., 1998,J. Virol., 72:436-441; D. M. Koelle et al., 1997, Human. Immunol.,53:195-205) were similarly derived from bulk cultures.

Some clones were derived using secondary in vitro stimulation withantigen. To derive additional TCC from donor 1 (D. M. Koelle et al.,1994, J. Virol., 68:2803-2810), PHS-driven bulk cultures were preparedfrom each of four 2 mm biopsies (day 5 of symptoms) obtained 6 yearsafter the recurrence from which clone 1A.B.25 (above) was derived. After16 days, 1.5×10⁶ bulk lymphocytes from one biopsy culture werestimulated with 10 μg/ml HSV-2 VP22 105-190 (see below) and an equalnumber of autologous irradiated (3300 rad) PBMC in 2 ml T-cell media (D.M. Koelle et al., 1994, J. Infect. Dis., 169:956-961). IL-2 (32 U/ml)was added starting on day 6. TCC 1.L3D5.10.8 was isolated from this lineon day 12 as described (D. M. Koelle et al., 1994, supra). To createPBMC-derived TCCSB.17 and BM.17, 1.5×10⁶ PBMC of HSV-2 seropositivedonors SB and BM were stimulated for 12 days with 4 μg/mlbaculovirus-derived full length VP16 in 25 well plates; responding cellswere cloned at 1 cell/well. TCC and lines were used 10-14 days afterlast stimulation.

All cell lines were negative for mycoplasma except ARENT. ARENT wasinitially positive for mycoplasma by DNA probe test (Genprobe, SanDiego, Calif.) and was treated with ciprofloxacin at 10 μg/ml (S. M.Gignac et al., 1991, Leukemia 5:162-165) for two weeks prior toutilization with conversion of the test to negative.

Flow Cytometry

A combination of murine mAb to human CD4 (clone SFCI 12T4D11) and CD8(clone SFC 21Thy2D3 recognizing the α chain of human CD8) (Coulter,Hialeah, Fla.) was used for flow cytometry.

Immunoblot

Lysates of HSV-infected Vero cells were prepared, electrophoresedthrough 10% SDS-PAGE gels, and transferred to nitrocellulose membrane asdescribed (R. A. Ashley et al., 1988, J. Clin. Microbiol. 26:662-667).Blots were blocked with PBS-0.05% Tween 20-1% nonfat dried milk. Antigenwas detected by sequential incubation with 1:100 dilution of mAb P43specific for the U_(L)49 gene product VP22 (G. D. Elliott et al., 1992,J. Gen. Virol., 73:723-736), affinity purified goat anti-mouseIgM-peroxidase conjugate (Sigma, St. Louis, Mo.), and TMB substratesystem (Kirkegaard and Perry, Gaithersberg, Md.) with washes (three×fiveminutes) in PBS-Tween between each step.

Viral DNA Libraries and Cloning

For subgenomic DNA, the HSV-2 strain HG-52 BamH I w fragment wassubcloned from the Bgl II i fragment and gel-purified. Viral DNA wasdigested with Sma I, BamH I ends were blunted with Klenow DNApolymerase, and DNA fragments were purified by phenol extraction andalcohol precipitation. For whole viral DNA, confluent Vero cells wereinfected with HSV-2 strain HG52. Total nucleic acids from three 150 cm²cell cultures were prepared by proteinase K digestion, chloroform-phenolextraction, and isopropanol precipitation. Resultant material wastreated with Range H and re-extracted and precipitated. Aliquots (1 μg)of HG52 DNA were digested with Sma I or Alu I and 80% of these digestswere combined and recovered as above for creation of expressionlibraries.

Expression cloning was performed using pUEX vectors (G. M. Bressan etal., 1987, Nucleic Acids Res., 15:10056). pUEX-1, -2, and -3 DNA waslinearized with Sma I, dephosphorylated with calf intestinalphosphatase, and gel purified. Approximately 100 ng of vector and 500 ngof DNA fragments were ligated and 10% of ethanol-precipitated reactionmixtures used to transform E. coli strain DH10 Electromas (GIBCO) byelectroporation (BTX, San Diego, Calif.) in 1 mm cuvettes. After onehour recovery in 1 ml SOC media, portions were frozen as glycerol stocks(100 μl each), tittered on ampicillin plates at 30° C. (250 μl), or useddirectly (250 μl) for protein induction to create fusion proteinlibraries. Several thousand ampicillin-resistant colonies were isolatedper transformation. To amplify genomic libraries, glycerol stocks weregrown overnight at 30° C. I n2YT-ampicillin and re-frozen.

Confirmatory subcloning of VP22 105-190, U_(L)50 118-312, and U_(L)50118-250 was performed by isolating the 262 base-pair Sma I-Stu Ifragment of U_(L)49, the 583 by Sma I fragment of U_(L)50, or the 397 bySma I-Stu I fragment of U_(L)50, respectively. Fragments were clonedinto the appropriate linearized, gel purified pUEX vector and proteinexpressed in E. coli DH5I. Constructs were confirmed by sequencing.

Antigens

Whole virus preparations containing 10⁸-10⁹ pfu/ml were UV-inactivatedfor 30 minutes (A. Mikloska and A. L. Cunningham, 1998, J. Gen. Virol.,79:353-361) and used at a 1:100 final dilution. Peptides of VP22 weresynthesized as described (D. M. Koelle et al., 1997, Human. Immunol.,53:195-205) and used as stocks at 2 mg/ml in DMSO. Peptides of U_(L)48,13 amino acids long and overlapping by four amino acids, VP16 of HSV-2amino acids 1-416, and full-length VP16, both expressed in baculovirus,were obtained from Chiron Corporation, Emeryville, Calif.

Bacterial-derived protein antigen expression was induced for two hoursat 42° C. in cells growing logarithmically (OD₆₀₀ 0.4-0.6) in2YT-ampicillin broth at 30° C. Protein was purified as described (P. I.Neophytou et al., 1996, Proc. Natl. Acad. Sci. USA, 93:2014-2018),omitting gel purification. Bacterial cultures of 50 ml (libraries) or5-10 ml cultures (pools and clones) yielded fine particulate suspensionsin 200-400 μl PBS (Ca, Mg-free). Protein concentrations were determinedby BCA (Pierce, Rockford, Ill.) after solubilizing proteins in 1% SDS at60° C. for 10 minutes. In some experiments, heat-induced bacteria werewashed with PBS and PBS/10 mM EDTA, heated to 56° C. for 10 minutes, andwashed in PBS prior to use as antigen.

After identification of an active library of viral DNA, antigenidentification used 30-60 clones for subgenomic viral DNA fragments or2,000-3,000 clones for full-length viral DNA. For the less complexlibrary, 1 ml cultures of each clone were processed as pools of six toeight clones. Individual clones within the active pool, and confirmatorysubclones containing known viral DNA fragments, were processed as 5 mlcultures. A combinatorial method (P. I. Neophytou et al., 1996, Proc.Natl. Acad. Sci. USA, 93:2014-2018) was used to screen libraries fromwhole viral DNA. Glycerol stocks of amplified libraries of transformedbacteria were tittered on ampicillin plates; 20-30 colonies/well werecultured overnight at 30° C. in a 96-well plate in a rotating shaker.Cultures were diluted 1:100 into 1 ml cultures and fusion proteinsynthesis induced as described above. Portions (400 μl) of cultures werepooled row- and column-wise for protein purification and evaluation inlymphoproliferation assays. If more than one row and column werepositive, wells at the intersections of positive rows and one positivecolumn were used to prepare protein from 5-10 ml cultures todefinitively identify a positive well. Cultures (n=96 colonies) ofbacteria were derived from ampicillin plates seeded with diluted brothfrom positive wells. These were evaluated as pools (of 12 bacterialcolonies) and then individual clones.

Lymphocyte Functional Assays

Triplicate proliferation assay wells contained 10⁴ cloned T-cells, 10⁵irradiated (3300 rad) PBMC or 2.5×10⁴ irradiated (8000 rad) EBV-LCL asantigen presenting cells (APC), and antigen in 200 μl T-cell media (D.M. Koelle et al., 1997, Human. Immunol., 53:195-205) in 96-well U-bottomplates. When heat-killed bacteria were used as antigen, the equivalentof 10⁵ cfu/well (prior to inactivation) was added and gentamicin (20μg/ml) was included. After 72 hours, 1 μCi/well ^([) ^(3])H thymidinewas added for 18 hours, cells were harvested, and incorporation ofthymidine evaluated by liquid scintillation counting. Standarddeviations were less than 10% of the mean values. Results are reportedas mean cpm or as delta cpm=mean cpm for experimental antigen minus meancpm for control antigen. Control antigen was mock-infected cell lysatefor whole viral antigens and pUEX2-derived β-galactosidase forrecombinant protein preparations. To determine the reactivity ofbulk-cultured lesion-derived T-cells, fusion proteins or controlβ-galactosidase were used at 10 μg/ml. Glycoproteins B and D and VP16 ofHSV-2 were used at 1 μg/ml and assays performed as previously described(D. M. Koelle et al., 1998, J. Clin. Invest., 101:1500-09). To determineHLA restricting loci, HLA DR-specific mAb L243 (V. G. Preston et al.,1978, J. Virol., 28:499-517), HLA DP-specific mAb B7.21 (A. J. Watson etal., 1983, Nature, 304:358-360), or HLA DQ-specific mAb SpV-L3 (H. Spitset al., 1984, Eur. J. Immunol., 14:299-304) were used as described (D.M. Koelle et al., 1994, J. Virol. 68:2803-2810).

Cytolysis assays were performed in triplicate using 4-hour ^([51])Crrelease as described (D. M. Koelle et al., 1993, J. Clin. Invest.,91:961-968). Target EBV-LCL were infected for 18 hours with HSV-3 at amultiplicity of infection of 30 or pulsed with 1.0 μM peptide for 90minutes prior to washing as described (W. W. Kwok et al., 1996, J. Exp.Med., 183:1253-1258). The effector to target ratio was 20:1. Spontaneousrelease was less than 28%.

DNA Sequencing

Viral inserts in plasmids in bacteria yielding active proteins werecompletely sequenced (Taq DyeDeoxy FS kit, Perkin-Elmer ABI, FosterCity, Calif.) in both directions starting with primersCATGGCTGAATATCGACGGT (SEQ ID NO: 1; 5′ end of insert) andCTAGAGCCGGATCGATCCGGTC (SEQ ID NO: 2; 3′ end of insert) and theninternal primers designed as required.

HLA Typing

HLA DR and DQ typing was performed at class II alleles by serologicmethods or at the DNA level by reverse dot blot hybridization (E.Mickelson et al., 1993, Tissue Antigens, 41:86-93). HLA DP typing wasperformed by sequencing (HLA DP kit, Perkin Elmer ABI).

Results

Fine Localization of T-cell Epitopes

To reduce the complexity of libraries for expression cloning, TCCrecognizing antigen(s) partially mapped using HSV-1×HSV-2 intertypicrecombinant viruses (IRV) were selected. HSV DNA near 0.7 map unitesencodes T-cell antigens in addition to VP16. Epitope mapping for TCC4.2EI and 2.3 (D. M. Koelle et al., 1994, J. Virol., 68:2803-2810) wasimproved with IRV DX32 (FIG. 1A). This HSV-2 based virus contains ablock of HSV-1 DNA near 0.7 map units (V. G. Preston et al., 1978, J.Virol., 28:499-517). The U_(L)48 gene product has the HSV-2 phenotype,as shown by reactivity with HSV-2 type-specific, VP16-specific (D. M.Koelle et al., 1994, J. Virol., 68:2803-2810) T-cell clone 1A.B.25. TheU_(L)49 (FIG. 2) and U_(L)50 gene products (M. V. Williams, 1987,Virology, 156:282-292; F. Wohlrab, 1982, J. Virol., 43:935-942) alsohave a HSV-2 phenotype. The HSV-2 DNA present in IRV DX32 thereforeincludes U_(L)48, U_(L)49, U_(L)50, and most likely the interveningU_(L)49.5. Since TCC 4.2E1 and 2.3 react with RS1G31 and DX32, but notwith RP2 (FIG. 1B), recognition of U_(L)49, U_(L)49.5, or U_(L)50 ismost likely.

Expression Cloning to Determine T-cell Antigens

The BamH I w fragment of HSV-2 was selected for expression cloning,since it contains the U_(L)49, U_(L)49.5, and most of the U_(L)50 codingsequences (A. Cress and S. J. Triezenberg, 1991, Gene, 103:235-238; G.D. Elliott and D. M. Meredith, 1992, J. Gen. Virol., 73:723-736; N. J.Maitland et al., 1982, Infect. Immun., 38:834-842). 70-90% of randomcolonies contained an insert; all were of viral origin. The most activelibraries (Table 1) for each TCC (pUEX1 for TCC 4.2E1, pUEX 3 for TCC2.3) were selected and an individual reactive bacterial clone detectedby sequential testing of pools and individual colonies (Table 2). Clone1.1.3 encodes a fusion protein eliciting proliferation by TCC 4.2E1.This clone contains a backwards 80 bp Sma I fragment of U_(L)49, a 262bp Sma I fragment of HSV-2 U_(L)49 DNA predicted to encode amino acids105 to 190, forward and in-frame with regards to β-galactosidase, and a246 bp Sma I fragment of U_(L)49 forward but out of frame due to adeletion of a single C residue at the 262 bp Sma I fragment-242 bp Sma Ifragment junction. Clone 3.19 contained a 583 bp Sma I fragment encodingamino acids 118-312 of U_(L)50, followed by backwards 80 and 96 bp Sma Ifragments of U_(L)49.

library¹ pUEX1-BamH I pUEX2-BamH I pUEX3-BamH I control stimuli² TCC“w”-Sma I “w”-Sma I “w”-Sma I media HSV-2 4.2E1 10,105 4,150 1,903 28621,591 2.3 418 785 2,279 102 11,014 library¹ pUEX1-HG52- pUEX2-HG52-pUEX3-HG52- control stimuli² TCC Sma I-Alu I Sma I-Alu I Sma I-Alu Imedia HSV-2 ESL4.9 −52 −25 16,235 146 66,013 ESL2.20 1 768 5,427 12313,359 ¹Library names list expression vector, name of HSV-2 restrictionfragment or strain of full-length viral DNA, and restriction enzyme(s)used to digest viral DNA. ²10⁵ autologous irradiated (3300 rad) PBMC andeither mock-infected cell lysate or UV-treated HSV-2 antigen.

Identification of T-cell antigens was confirmed by targeted subcloningand overlapping peptides. The 262 bp Sma I fragment of U_(L)49 of HSV-2encoding amino acids 105-190 was subcloned into pUEX3 to yield plasmid49.262.12. This protein stimulated TCC 4.2E1 (Table 2). Only peptide105-126 of VP22 of HSV-2 (GGPVGAGGRSHAPPARTPKMTR; SEQ ID NO: 3) wasstimulatory FIG. 3). DNA fragments encoding U_(L)50 118-312 and 118-250were subcloned into pUEX3. Fusion proteins expressing these fragmentswere active (Table 2).

TABLE 2 Antigenic specificity of HSV-2 reactive TCC. Bacterially-derivedrecombinant fusion protein antigens were used at 1:900 dilution.Autologous EBV-LCL (clone 4.2E1) or PBMC were used as APC. Data aredelta cpm [³H] thymidine incorporation compared to media, which was lessthan 500 cpm in each case. recombinant antigen control antigens TCCClone name viral sequence¹ cpm pUEX2 β-gal HSV-1 HSV-2 4.2E1 1.1.3 VP22105-190  4,875 93 nd nd 49.262.12² VP22 105-190  6,898 2.3 3.19 U_(L)50118-312 43,971 231 543 53,032 50.583.44³ U_(L)50 118-312 34,453 50.397³U_(L)50 118-250 66,501 ESL4.9 C11 VP22 177-220 59,400 166 112,803 64,685ESL2.20 C9D10 U_(L)21 148-181 23,543 173 0 37,989 ¹Amino acids predictedforward and in-frame with β-galactosidase from sequence data.²Confirmatory subclone of 1.1.3 containing only a 262 bp Sma I fragmentof U_(L)49 DNA forward and in-frame with pUEX3. ³Confirmatory subclonesof 3.19 containing a 583 bp Sma I fragment of ULSO or a 397 bp Sma I-StuI fragment of U_(L)50 DNA forward and in-frame with pUEX3.

Evaluation of random colonies from full-length HSV-2 DNA librariesshowed that 80-100% contained plasmids with an insert; 80-100% ofinserts were of viral origin. For both TCC ESL4.9 and ESL2.20, only thepUEX3 protein library elicited lymphoproliferation (Table 1). Since thelibraries were more complex than for those made from the BamH I wfragment, 2,000-3,000 bacterial transformants were screened by acombinatorial method. In preliminary experiments, heat-killed, washedbacteria were found to substitute for inclusion body preparations ofprotein in lymphoproliferation assays at the pool (5-12 bacterialclones) and final assay stages.

Sequencing of plasmids in positive bacteria showed that TCC ESL4.9recognized a 44 amino acid fragment of U_(L)49 gene product VP22 (aminoacids 177-220), while TCC ESL2.20 recognized a 34 amino acid fragment ofU_(L)21 (amino acids 148-181) (Table 2). In both cases single Alu Ifragments of HSV-2 DNA were inserted in-frame and forwards. Peptidemapping revealed that amino acids 187-206 (FIG. 3C) stimulated TCCESL4.9.

Fusion Proteins as Probes of Bulk Lesion-infiltrating T-cells

Newly discovered T-cell antigens were added to the panel of HSV antigensused to probe bulk cultures of lesion-infiltrating T-cells. The firstavailable specimens were a set of four biopsies (2 mm each) obtainedfrom day 5 (virus culture positive) of a buttock recurrence of HSV-2from patient 1 (D. M. Koelle et al., 1998, J. Clin. Invest. 101:1500-09;D. M. Koelle et al., 1994, J. Virol., 68:2803-2810). All four biopsiesshowed reactivity with VP22 105-190 but not β-galactosidase,glycoproteins B or D, or VP16. TCC were derived after restimulating theoriginal bulk culture for one cycle with VP22 105-190 fusion protein.Proliferative responses of TCC 1.L3D5.10.8 (FIG. 3B) to VP22 (105-190)and constituent peptides document a third T-cell epitope in VP22contained within amino acids 125-146.

Definition of Additional T-cell Epitopes in Tegument protein VP16

Three epitopes within VP16 (Table 3), all HSV-2 type-specific werepreviously identified (K. R. Jerome et al., 1998, J. Virol.,72:436-441), and proliferative responses to full-length VP16 in bulkcultures of genital HSV-2 lesion-infiltrating lymphocytes from four ofseven (57%) patients were detected (D. M. Koelle et al., 1998, J. Clin.Invest., 101:1500-09). Additional peptide epitopes were sought withinVP16 by two strategies. The first strategy involved screening panels ofclones recovered from lesion vesicle fluid for reactivity withrecombinant VP16 of HSV-2 followed by epitope mapping with peptides.Peptides containing amino acids 185-197 and the overlapping pair 209-221and 213-225 were stimulatory for TCC RH.13 and KM.7, respectively (Table3). All other VP16 peptides were negative (<500 cpm). The secondstrategy involved using PBMC as starting material and secondary in vitrostimulation with recombinant baculovirus-derived VP16. Clones BM.17 andSB.17) from two individuals recognized the same peptide (amino acids437-449) as well as β-gal-VP16 fusion protein and whole virus. All threenewly defined VP16 epitopes were type-common, shared by HSV-1 and HSV-2whole virus preparations, as expected from sequence data (A. Cress andS. J. Triezenberg, 1991, Gene, 103:235-238).

TABLE 3 Epitopes within VP16 of HSV-2 recognized by lesion- andPBMC-derived CD4 TCC. Data are delta cpm [³H] thymidine incorporationcompared to media, which was less than 500 cpm in each case. recombinantHSV-2 whole protein¹ TCC virus antigen VP16 β-gal-BP16 HSV-2 VP16peptide name origin HSV-1 HSV-2 1-492 180-492 amino acids delta cpmnewly reported epitopes RH.13 lesion 3,340 3,407 32,991 nd 185-19755,614 KM.7 lesion 6,093 5,847 5,627 nd 209-221 10,075 BM.17 PBMC 30,78413,777 nd 45,958 437-449 79,723 SB.17 PBMC 2,207 4,187 nd 12,178 437-44936,442 previously reported epitopes ESL4.34 lesion 256 8,780 17,302 nd389-401 12,968 393-405 95,942 ESL3.334 lesion 253 14,232 22,754 16,434430-444 27,283 1A.B.25 lesion 414 33.493 24,919 41,123 431-440 38,664¹VP16 1-492 (baculovirus-derived) was used at 1 μg/ml. β-gal-BP16180-492 was used at 1:1,000 dilution. ²Peptides were used at 1 μM. na =not available nd = not done

HLA Restriction

The HLA restriction of the TCC recognizing antigens encoded near 0.7 mapunits was determined in detail. Proliferation of TCC 4.2E1, specific forVP22 105-126, is inhibited 84% by anti-DP, but less than 20% by anti-DRor anti-DQ mAb. TCC 4.2E1 is from a DPB1*2001/DPB1*0402 heterozygousdonor. Allogeneic EBV-LCL bearing DPB1*2001, but not DPB1*0402, presentantigen (Table 4), establishing restriction by DPB1*2001. Proliferationof TCC 2.3, specific for U_(L)50, was inhibited by anti-DR but notanti-DP or anti-DQ mAb. This clone is from a DRB1*0301/BRB1*0701heterozygous donor. Allogeneic PBMC from a DRB1*0301 donor presentedantigen, consistent with binding of antigenic peptide to this allele.However, presentation by the linked DR gene products DRw52 or DRw53,cannot be ruled out. Additional HLA restriction studies are summarizedin Table 5.

TABLE 4 Determination of restricting HLA allele of lesion-derived CD4TCC 4.2E1 and 2.3. Antigens were β-gal fusion proteins (Table 2) at1:900 dilution. Data are delta cpm [³H] thymidine incorporation comparedto media, which was less than 500 cpm in each case. T-cell clone antigenAPC HLA type¹ delta cpm² 4.2E1 1.1.3 autologous EBV- DPB1*0402, 200130,719 LCL AMAI EBV-LCL DPB1*0402 2,732 ARENT EBV-LCL DPB1*2001 26,2182.3 50.583.44 autologous PBMC DRB1*0301, 0701 8,964 allogeneic PBMC ADRB1*0701, 1001 45 allogeneic PBMC B DRB1*0301, 1301 19,223 ¹HLA type atthe HLA class II locus as determined by inhibition with mAb. ²Incomparison to pUEX2 control protein (1:1000 dilution) with the same APC,which caused less than 500 cpm [³H] thymidine incorporation in eachcase.

TABLE 5 Cytolytic activity of lesion-derived, tegument-specific CD4 TCCwith summary of fine specificity and HLA restriction. Results arepercent specific release at an effector to target ratio of 20:1 exceptESL4.34 (10:1). Auto = autologous EBV-LCL as target cells; allo =allogeneic EBV-LCL mismatched at the relevant HLA locus (if known) ormismatch at HLA DR and DQ. cytolysis assay target HLA auto auto autoallo allo allo TCC specificity¹ restriction² HSV-2 peptide mock HSV-2peptide mock newly reported epitopes 4.2E1 VP22 105-126 DPB1*2001 20.744.2 −4.1 −2.9 −1.7 4.6 ESL4.9 VP22 187-206 DR³ −0.6 20.2 1.3 0 0 0ESL2.20 U_(L)21 148-181 DR³ 2.7 na 0.9 0 na 0 1.L3D5.10.8 VP22 125-146DR⁴ 1.1 61.1 −0.3 −0.4 −0.6 −0.4 1.L3D5.10.12 VP22 125-146 DR⁴ 2.5 57.61.6 −0.1 −2.5 −1.4 RH.13 VP16 185-197 DR⁴ 62.5 55.2 −0.9 9.6 0.3 1.8KM.7 VP16 209-221 DR⁴ 38.7 43.6 2.7 −2.2 4.3 −1.1 BM.17 VP16 437-449DQB1*0501 10.1 28.5 −0.3 nd nd nd SB.17 VP16 437-449 DQB1*0501 48.7 60.65.4 nd nd nd 2.3 U_(L)50 118-250 DRB1*0301 0.8 na 0 1.1 na 0 previouslydescribed epitopes ESL4.34 VP16 393-405 DRB1*0402 2.1 10.4 1.0 0.5 0.60.3 BSL3.334 VP16 430-444 DQB1*0302 12.3 33.6 0.7 1.4 0.3 2.2 1A.B.25VP16 431-440 DQB1*0201 24.3 42.2 1.9 1.7 2.1 −0.4 na = not availablesince epitope mapping was not done and synthetic antigenic peptide wasnot made. nd = not done.

The HLA restriction of TCC BM.17 was studied in detail. Proliferation ofTCC BM.17 and the similar clone SB.17 was inhibited 90% by anti-DQ, butless than 25% by anti-DR or anti-DP mAb. Donors BM and SB areheterozygous for HLA DQB1*0201/0501. At high concentrations of peptide,both DQB1*0201- and DQB1*0501 homozygous EBV-LCL appeared to presentantigen to TCC BM.17. However, DQB1*0501 homozygous cells presentedpeptide much more efficiently than DQB1*0201 homozygous cells (FIG. 4).Thus, three different but overlapping epitopes in VP16 431-449 arepresented by HLA DQB1*0302, DQB1*0201, and DQB1*0501.

CTL Activity of Tegument-specific CD4 T-cell Clones

Cytotoxic activities of the CD4 TCC with newly and previously identifiedspecificities were tested using EBV-LCL target cells (Table 5). Allclones tested displayed cytolytic activity towards peptide-loaded targetcells. Cytolytic activity against target cells infected with HSV-2showed greater variability. VP22-specific TCC 4.2E 1 was active, whileVP22-specific TCC from other donors were not. Among the sevenVP16-specific T-cell clones tested, six displayed greater than 10%cytotoxicity towards HSV-2-infected target cells. The single U_(L)21-and U_(L)50-specific TCC were not active against virally-infected targetcells.

Discussion

HSV-specific T-cells selectively infiltrate recurrent genital HSV-2lesions (D. M. Koelle et al., 1994, J. Infect. Dis., 169:956-961). LocalCTL activity, with CD4 and CD8-mediated components, is correlated withviral clearance (D. M. Koelle et al., 1998, J. Clin. Invest.101:1500-09). The antigens recognized by local HSV-specific T cells arediverse and in many cases unknown (D. M. Koelle et al., 1994, J. Virol.,68:2803-2810). This example documents recognition of tegument proteinsVP22 and U_(L)21 and the viral dUTPase, and provides new informationabout tegument protein VP16.

The expression cloning system described herein works well with HSV.Genomic double stranded DNA can be used directly since introns are rarein the HSV genome. The same HSV-2 strain, HG52 (A. Dolan et al., 1998,J. Virol. 72:2010-2021) was used to screen candidate lesion-derived TCCand make protein libraries. The relatively low degree of strainvariability (M. J. Novotny et al., 1996, Virology, 221:1-13) betweenHSV-2 strains in the donors and HG52 might rarely lead to omission ofepitope(s) recognized in vivo; application to viruses with more strainvariation would benefit from the use of autologous isolates.

Notably, reactivity with VP22 was detected in two independent expressioncloning experiments with lesion-infiltrating TCC from two donors. VP22reactivity was also detected during screening of the first available setof bulk lesion-infiltrating lymphocyte cultures. Ten additional clonesfrom three patients have been negative with the disclosed fragments ofU_(L)49, U_(L)21, and U_(L)50.

Tegument antigens may be suitable targets for lesion-infiltrating CD4T-cells because of their abundance. VP16 and VP22 are present in largeamounts: on the order of 1.6×10³ molecules of VP16 (Y. Zhang and J. L.C. McKnight, 1993, J. Virol., 67:1482-1492) and 2.5-2.8×10³ molecules ofVP22 (J. Leslie et al., 1996, Virology, 220:60-68) are incorporated intoeach virion in HSV-1. Less information is available for U_(L)21 (J. D.Baines et al., 1994, J. Virol. 68:2929-2936; J. A. Blaho et al., 1994,J. Biol. Chem. 269:17401-17410). The viral dUTPase is the firstnon-virion component documented to be a target of the HSV-specific CD4T-cell response. This enzyme, like VP16 and VP22, localizes to thenucleus of HSV-2 (although not HSV-1) infected cells (F. Wohlrab et al.,1982, J. Virol., 43:935-942). Antigen presentation in vivo may occurafter endogenous synthesis of dUTPase in infected cells, or scavengingof dUTPase antigen from infected cell debris. Lysis of HSV-infectedcells by dUTPase-specific TCC 4.2E1 indicates that, at least in vitro,presentation of endogenous antigen can occur.

Because polypeptides expressed as C-terminal fusion to VP22 can beco-transported into cells, expression of proteins as VP22 fusions may beof interest as a type of adjuvant preparation. This can be tested byexpression of heterologous epitopes in VP22. VP16 and VP22 of HSV-1 arestrongly, noncovalently associated in infected cells as shown bycoimmunoprecipitation. These proteins co-localize in the perinucleararea of cells (G. Elliott et al., 1995, J. Virol., 69:7932-7941; G. D.Elliott et al., 1992, J. Gen. Virol., 73:723-736). This association mayplay a role in stimulating the apparent high level of CD4 T-cellresponse to VP16.

In summary, expression cloning has allowed discovery of novel HSV T-cellantigens. The in situ enrichment of antigen-specific CD4 T-cells inlesions allows study of the antigenic repertoire unbiased by secondaryin vitro stimulation with antigen. The favorable characteristics of theHSV genome allow direct use of libraries of whole viral DNA. Tegumentproteins are candidates together with membrane glycoproteins for use asHSV vaccines in humans.

Example 2 Identification of Additional HSV-2 Viral Epitopes

The expression cloning method described in Example 1 above was employedto identify additional T cell antigens of HSV-2. The results revealedtwo additional antigens. One is found at amino acids 1078-1319 ofU_(L)19. U_(L)19 is also known as major capsid antigen or as VP5. Theother antigen is amino acids 1-273 of US8, also known as glycoprotein E.The US8 antigen was identified using T cells derived from a cervicalsample.

Example 3 Efficacy of Full-length U_(L)49 and U_(L)50

This Example shows that the full-length U_(L)49 and U_(L)50 proteins areeffective at stimulating T cell proliferation. The data demonstrate theantigenicity of full-length U_(L)49 expressed in E. coli and in Cos-7cells, and the antigenicity of full-length U_(L)50 expressed in Cos-7cells. These results confirm that the antigens described hereinabovewere accurately identified.

To express full-length U_(L)49 protein of HSV-2 in a prokaryotic system,the gene was cloned by PCR from DNA prepared from HSV type 2 strain HG52using primers GGAAGATCTACCTCTCGCCGCTCCGTCA (SEQ ID NO: 4) at the 5′ endof the gene and CCGGAATTCTTGTCTGTCGTCTGAACGCG (SEQ ID NO: 5) at the 3′end of the gene. PCR product was digested with Bgl II and EcoR I andcloned into the Bgl II and EcoR I sites in the TA cloning vectorpcR2.1-Topo (Invitrogen). The gene was then subcloned into the vectorpTrcHisB (Invitrogen) and then into pGEX-2T (Pharmacia). The sequence ofthe HSV-2 U_(L)49 clone had one coding mutation compared to thepublished sequence (Dolan 1998): amino acid 244 was mutated from serineto proline. The predicted amino acid sequence of the expressed proteinalso is missing the initial methionine. U_(L)49 contains an N-terminalfusion domain derived from vector pGEX2T. This plasmid is namedpGEX2T-U_(L)49HSV2.

To make prokaryotically expressed full length U_(L)49 of HSV-2,pGEX2TU-L49HSV2 or control empty vector was transformed into E. colistrain BL21 Bacteria in log-phase growth were adjusted to an OD₆₀₀ of0.4 in LB-ampicillin media. To some tubes isopropylbeta-D-thiogalactopyranoside (IPTG) was added to 0.3 mM. Bacteria werecultured for 1.5 hours at 37° C. with rotation. Bacteria were collectedby centrifugation and washed 3× in PBS containing 1 mM EDTA, heated to65° C. for 10 minutes, and washed twice more with PBS, and resuspendedat approximately 1×10⁹ bacteria/ml in T-cell medium. Heat-killedbacterial suspensions were used as test antigen.

To express full-length U_(L)49 protein of HSV-2 in a eukaryotic system,the gene was separately re-amplified by polymerase chain reaction usinga high-fidelity DNA polymerase with proof-reading function. The sameprimers and template were used. The gene was cloned directly into theBgl II and EcoR I sites of pEGFP-C1 (Clontech). The entire U_(L)49 genewas sequenced and agreed with published sequence. The predicted aminoacid sequence of the expressed protein is identical to that predictedfor viral U_(L)49 except that the initial methionine at amino acid 1 ismissing. A N-terminal fusion domain derived from vector pEGFP-C1 is alsopredicted to be expressed. This plasmid is named pEGFP-C1-UL49HSV2.

To make eukaryotically expressed full length U_(L)49 of HSV-2,pEGFP-C1-UL49HSV2 plasmid DNA or pEGFP-C1vector control DNA wastransfected into Cos-7 cells by lipofection. After 48 hours, cells werescraped and sonicated and a supernatant and pellet phase prepared. Cellsfrom a 9.4 cm² dish were used to prepare 300 microliters of supernatant.The pellet from a 9.4 cm² dish was resuspended in 300 microlitersmedium. Supernatant and pellet preparations were used as test antigens.

To express full-length UL50 protein of HSV-2 in a eukaryotic system, thegene was cloned by PCR using high-fidelity thermostable DNA polymerasewith proof-reading function from DNA prepared from HSV type 2 strainHG52 DNA using primers TAAGGTACCTATGAGTCAGTGGGGGCCC (SEQ ID NO: 6) atthe 5′ end of the gene and AAACTGCAGGAGGCGCGGTCTAGATGC (SEQ ID NO: 7) atthe 3′ end of the gene. The target DNA was used as a clone of the Bgl IIi fragment cloned into pUC9. The PCR product was digested with Kpn I andPst I and cloned into similarly digested pcDNA3.1-myc-his-B(Invitrogen). The sequence was confirmed at the junctions between vectorand insert. The plasmid is named pcDNA3.1-myc-his-B-UL50HSV2. Thepredicted amino acid sequence of the expressed protein is identical tothat predicted for viral U_(L)50. A N-terminal fusion domain derivedfrom vector pcDNA3.1-myc-his-B is also predicted to be expressed. Tomake eukaryotically expressed full-length U_(L)50 of HSV-2 as testantigens, the Cos-7 system was used exactly as described above forU_(L)49. Control antigen for U_(L)50 was made by transfecting Cos-7cells with pcDNA3.1-myc-his-B.

These test antigens were added to assay wells (96-well, U-bottom) in 200microliters of T-cell medium containing 1×10⁵ autologous irradiatedperipheral blood mononuclear cells (PBMC) per well and 1×10⁴lesion-derived CD4-bearing T-cell clone ESL4.9 for U_(L)49 or clone 2.3for U_(L)50 (Koelle et al, 1994 and 1998 and original patentapplication). Assays were performed in duplicate or triplicate. Afterthree days, ³H thymidine incorporation was measured as described inExample 1.

Results are expressed as stimulation index (mean cpm ³H thymidineincorporation test antigen/mean cpm ³H thymidine incorporation mediacontrol) and delta cpm (mean cpm ³H thymidine incorporation test antigenminus mean cpm ³H thymidine incorporation media control). Positive andnegative control antigens were run as indicated and as described inExample 1.

TABLE 6 Antigenicity of full-length HSV-2 UL49 expressed prokaryoticallyin E. coli BL21 final antigen dilution delta cpm stimulation index UVHSV-2 1:100 26,823 386 heat-killed pGEX2 1:4 −11 0.84 heat-killed pGEX21:40 −25 0.64 heat-killed pGEX2 1:400 −8 0.89 heat-killed pGEX2- 1:49,413 135 UL49HSV2 heat-killed pGEX2- 1:40 10,526 152 UL49HSV2heat-killed pGEX2- 1:400 5,021 73 UL49HSV2

TABLE 7 Antigenicity of full-length HSV-2 UL49 expressed eukaryoticallyin Cos-7 cells stimulation antigen final dilution delta CPM indexUV-mock virus 1:100 −4 0.96 UV HSV-2 1:100 46,510 470 supernatant ofcontrol- 1:4 8 1.08 transfected cells pellet of control-transfected 1:4131 2.32 cells supernatant of UL49- 1:4 1,512 16.3 transfected cellspellet of UL49-transfected cells 1:4 84,951 859 pellet ofUL49-transfected cells 1:40 35,753 362 pellet of UL49-transfected cells1:400 29,854 302

TABLE 8 Antigenicity of full-length HSV-2 UL50 expressed eukaryoticallyin Cos-7 cells stimulation antigen final dilution delta CPM indexUV-mock virus 1:100 −43 0.89 UV HSV-2 1:100 52,990 135 supernatant ofcontrol-transfected 1:5 302 1.86 cells pellet of control-transfectedcells 1:5 34 1.09 supernatant of UL50-transfected 1:5 26,910 77.7 cellssupernatant of UL50-transfected 1:20 33,063 95.2 cells supernatant ofUL50-transfected 1:100 20,438 59.2 cells supernatant of UL50-transfected1:500 2,346 7.7 cells pellet of UL50-transfected cells 1:5 42,820 123.0pellet of UL50-transfected cells 1:20 18,487 53.7 pellet ofUL50-transfected cells 1:100 8,947 26.5 pellet of UL50-transfected cells1:500 864 3.5

These results show that HSV-2 proteins U_(L)49 and U_(L)50 retain theirimmunogenicity when expressed as full-length proteins. U_(L)49 wasstudied in prokaryotic and eukaryotic systems and U_(L)50 in aeukaryotic system.

Example 4 Efficacy of Full-length U_(L)21

To express full-length U_(L)21 protein of HSV-2 in a eukaryotic system,the gene was cloned by PCR using high-fidelity thermostable DNApolymerase with proof-reading function from DNA prepared from HSV type 2strain HG52 DNA using primers CTGGGATCCATGGAGCTCAGCTATGCCACC (SEQ ID NO:8) at the 5′ end of the gene and CGCGAATTCTCACACAGACTGGCCGTGCTG (SEQ IDNO: 9) at the 3′ end of the gene. The PCR product was digested with BamHI and EcoR I and cloned into similarly digested pGEX-5T. From there, itwas cut out with BamH I and Xho I and cloned into similarly digestedpcDNA3.1-myc-his-C (Invitrogen). The sequence was confirmed at thejunctions between vector and insert. The plasmid is namedpcDNA3.1-myc-his-C-UL21HSV2. The predicted amino acid sequence of theexpressed protein is identical to that predicted for viral U_(L)21. AN-terminal fusion domain derived from vector pcDNA3.1-myc-his-B is alsopredicted to be expressed. To make eukaryotically expressed full-lengthU_(L)21 of HSV-2 as test antigens, the Cos-7 system was used exactly asdescribed for U_(L)49. Control antigen for U_(L)21 was made bytransfecting Cos-7 cells with pcDNA3.1-myc-his-B.

The U_(L)21 test antigens were added to assay wells (96-well, U-bottom)in 200 microliters of T-cell medium containing 1×10⁵ autologousirradiated peripheral blood mononuclear cells (PBMC) per well and 1×10⁴lesion-derived CD4-bearing T-cell clone ESL2.20 (Koelle et al, 1994 and1998 and Example 1 above). Assays were performed in triplicate. Afterthree days, ³H thymidine incorporation was measured as described inExample 1. Results are expressed as stimulation index (mean cpm ³Hthymidine incorporation test antigen/mean cpm ³H thymidine incorporationmedia control) and delta cpm (mean cpm ³H thymidine incorporation testantigen minus mean cpm ³H thymidine incorporation media control).Positive and negative control antigens were run as indicated; details ofwhich can be found in Example 1. Results are presented in Table 9.

TABLE 9 Antigenicity of full-length HSV-2 UL21 expressed eukaryoticallyin Cos-7 cells.. stimulation antigen final dilution delta CPM indexUV-mock virus 1:100 43 1.75 UV HSV-2 1:100 5620 97.9 supernatant ofcontrol- 1:20 −9 0.83 transfected cells pellet of control-transfected1:20 −9 0.83 cells supernatant of UL21- 1:20 1870 33.25 transfectedcells supernatant of UL21- 1:100 3242 56.9 transfected cells supernatantof UL21- 1:500 4472 78.11 transfected cells supernatant of UL21- 1:20002526 46.79 transfected cells pellet of UL21-transfected cells 1:20 360663.24

Example 4 Prevalence of Antigens in Population

This example supports the utility of preventative and therapeutic usesof the antigens of the invention by demonstrating the prevalence ofresponses to these antigens among the population. To do this, sevenindividuals who were HSV-2 infected as documented by type-specificserology were surveyed. These individuals were different from theindividuals from whom the index T-cell clones were recovered from HSV-2lesions.

For each subject, PBMC were isolated and plated at 2×10⁶ cells/well in 2mls of T-cell medium in 24-well plates and stimulated in vitro with a1:500 dilution of UV-inactivated HSV-2 strain 333 for five days. At thattime, 40 units/ml recombinant human IL-2 was added for an additionalfive to six days, giving rise to a short-term, HSV-specific cell linetermed a B1 cell line.

Reactivity to individual HSV-2 proteins was assessed as follows.Proliferation assays were set up on 96-well round bottom microtiterplates, and each condition was performed in triplicate. To each well,1×10⁵ autologous irradiated (3300 rad gamma) PBMC were added as antigenpresenting cells. To each well, 1×10⁴ B1 cells were added. The followingcontrol substances were added: media, UV-treated mock virus preparationdiluted 1:500, UV-treated HSV-2 strain 333 diluted 1:500, glycoproteinsB or D or VP16 protein of HSV-2 (purified) at 4 micrograms per ml finalconcentration. The response to UV-treated HSV-2 was expected to bepositive and served as a positive control for the viability and overallspecificity of the cells. Glycoproteins B and D and VP16 were previouslyshown to be targets of HSV-specific T-cells (D. M. Koelle et al., 1994,J. Virol 68(5):2803-2810).

For the newly discovered antigens UL21, UL49, UL50, the cloning of thefull-length genes and their expression in the eukaryotic Cos-7 systemwas as described above, as was the preparation of control antigens basedon the empty vector. For the newly discovered antigen gE2 (US8), thefull-length gene was cloned with high-fidelity thermostable DNApolymerase with proof-reading function from DNA prepared from HSV type 2strain HG52 DNA using primers CGGGGTACCTGCTCGCGGGGCCGGGTTGGTG (SEQ IDNO: 10) at the 5′ end of the gene and TGCTCTAGAGCCTTACCAGCGGACGGACGG(SEQ ID NO: 11) at the 3′ end of the gene. The PCR product was digestedwith ACC65 I and Xba I and cloned into similarly digestedpcDNA3.1-myc-his-B (Invitrogen). The plasmid is namedpcDNA3.1-myc-his-B-US8. The sequence was confirmed at the junctionsbetween vector and insert. The predicted amino acid sequence of theexpressed protein is identical to that predicted for viral US8. AN-terminal fusion domain derived from vector pcDNA3.1-myc-his- is alsopredicted to be expressed. To make eukaryotically expressed full-lengthUS8 of HSV-2, the Cos-7 system was used as described above. For each ofthe four new antigens (UL21, UL49, UL50, and US8) and control, thesupernatant and pellet after sonication of transfected Cos-7 cells wasused at a final dilution of 1:20 in triplicate proliferation assays.

Positive responses were scored if the stimulation index (mean cpm ³Hthymidine incorporation for test antigen/mean cpm ³H thymidineincorporation for relevant control antigen) was greater than or equal to4.0. For UV HSV-2 antigen, the relevant control antigen was UV-mockvirus. For gB2, gD2, and VP16, the control was media. For the newantigens expressed in Cos-7 cells, the control antigen was either thepellet or supernatant of Cos-7 cells transfected with control emptyvector. Results are shown in Table 10. Reactivity with each of the newlydiscovered antigens was documented in at least one study subject.Overall, reactivity with UL49 was observed more frequently and similarto that for the known antigens gB2 and gD2. These data provide supportthat human individuals, in addition to the index subjects in whom theT-cell reactivity was originally described, are capable of reacting tothese antigenic HSV-derived proteins.

TABLE 10 Antigenicity of known and of newly discovered HSV-2 antigensamong a group of seven randomly chosen HSV-2 infected immunocompetentadults. ANTIGEN VP16 of UL49 of UL50 of UL21 of US8 of HSV-2 gB2 gD2HSV-2 HSV-2 HSV-2 HSV-2 HSV-2 n  7  5  5 0  5  1  1  2 % 100 71 71 0 7114 14 28

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

11 1 20 DNA herpes simplex virus 1 catggctgaa tatcgacggt 20 2 22 DNAherpes simplex virus 2 ctagagccgg atcgatccgg tc 22 3 22 PRT herpessimplex virus 3 Gly Gly Pro Val Gly Ala Gly Gly Arg Ser His Ala Pro ProAla rg 1 5 10 15 Thr Pro Lys Met Thr Arg 20 4 28 DNA herpes simplexvirus 4 ggaagatcta cctctcgccg ctccgtca 28 5 29 DNA herpes simplex virus5 ccggaattct tgtctgtcgt ctgaacgcg 29 6 28 DNA herpes simplex virus 6taaggtacct atgagtcagt gggggccc 28 7 27 DNA herpes simplex virus 7aaactgcagg aggcgcggtc tagatgc 27 8 30 DNA herpes simplex virus 8ctgggatcca tggagctcag ctatgccacc 30 9 30 DNA herpes simplex virus 9cgcgaattct cacacagact ggccgtgctg 30 10 31 DNA herpes simplex virus 10cggggtacct gctcgcgggg ccgggttggt g 31 11 30 DNA herpes simplex virus 11tgctctagag ccttaccagc ggacggacgg 30

What is claimed is:
 1. A pharmaceutical composition comprising anisolated HSV polypeptide and a pharmaceutically acceptable carrier,wherein the polypeptide comprises an amino acid sequence selected fromthe group consisting of: amino acids 18-312 and 118-250 of U_(L)50. 2.The composition of claim 1, wherein the polypeptide is a fusion protein.3. The composition of claim 2, wherein the fusion protein is soluble. 4.The pharmaceutical composition of claim 1, further comprising anadjuvant.
 5. A pharmaceutical composition comprising an isolated herpessimplex virus (HSV) polypeptide, wherein the polypeptide comprises aU_(L)50 protein, and a pharmaceutically acceptable carrier.
 6. Thecomposition of claim 5, wherein the polypeptide is a fusion protein. 7.The composition of claim 6, wherein the fusion protein is soluble. 8.The pharmaceutical composition of claim 5, further comprising anadjuvant.
 9. A method of treating or preventing an HSV infection in asubject comprising administering the composition of claim 5 to thesubject.
 10. A method of enhancing proliferation of HSV-specific T cellscomprising contacting the HSV-specific T cells with an isolatedpolypeptide that comprises a U_(L)50 polypeptide or immunogenic fragmentthereof.
 11. The method of claim 10, wherein the immunogenic fragmentcomprises amino acids 118-250 or 118-312 of U_(L)50.
 12. A method ofenhancing the production of HSV-specific antibodies in a subjectcomprising administering to the subject an isolated polypeptide thatcomprises a U_(L)50 polypeptide or immunogenic fragment thereof.
 13. Themethod of claim 12, wherein the immunogenic fragment comprises aminoacids 118-250 or 118-312 of U_(L)50.
 14. A polynucleotide that encodes apolypeptide comprises an amino acid sequence consisting essentially of:amino acids 118-312 or amino acids 118-250 of U_(L)50.
 15. A vectorcomprising the polynucleotide of claim
 14. 16. A host cell transformedwith the vector of claim
 15. 17. A method of producing an HSVpolypeptide comprising culturing the host cell of claim 16 andrecovering the polypeptide so produced.
 18. An HSV polypeptide producedby culturing a host cell transformed with a vector comprising apolynucleotide encoding an HSV U_(L)50 polypeptide wherein the HSVU_(L)50 polypeptide consists essentially of amino acids 118-250 or118-312 of U_(L)50.
 19. A recombinant virus genetically modified toexpress the polypeptide of claim
 18. 20. A fusion protein comprising anHSV polypeptide fused to a heterologous polypeptide, wherein the HSVpolypeptide consists essentially of amino acids 118-250 or 118-312 ofU_(L)50.
 21. A fusion of claim 20 that is soluble.
 22. A polynucleotidethat encodes a fusion protein of claim
 20. 23. A vector comprising thepolynucleotide of claim
 22. 24. A host cell transformed with the vectorof claim
 23. 25. A method of producing a fusion protein comprisingculturing the host cell of claim 24 and recovering the fusion protein soproduced.
 26. A fusion protein produced by the method of claim
 25. 27. Afusion protein of claim 26 that is soluble.
 28. A pharmaceuticalcomposition comprising the fusion protein of claim 26, and apharmaceutically acceptable carrier.
 29. The pharmaceutical compositionof claim 28, further comprising adjuvant.
 30. A pharmaceuticalcomposition comprising the fusion protein of claim 20, and apharmaceutical acceptable carrier.
 31. The pharmaceutical composition ofclaim 30, further comprising an adjuvant.
 32. A recombinant virusgenetically modified to express a U_(L)50 protein wherein the proteinconsists essentially of amino acids 118-250 or 118-312 of U_(L)50. 33.The recombinant virus of claim 32 which is a vaccinia virus, canary poxvirus, lentivirus, retrovirus, herpes virus or adenovirus.
 34. Apharmaceutical composition comprising the virus of claim 33 and apharmaceutically acceptable carrier, wherein the virus is a vaccininiavirus or acanary pox virus.
 35. The pharmaceutical composition of claim34, further comprising an adjuvant.
 36. A recombinant non-HSV virusgenetically modified to express a U_(L)50 protein.
 37. A recombinantnon-HSV virus of claim 36 which is a vaccinia virus, canary pox virus,lentivirus, retrovirus, herpes virus or adenovirus.
 38. A pharmaceuticalcomposition comprising the non-HSV virus of claim 36 and apharmaceutically acceptable carrier, wherein the virus is a vacciniavirus or a canary pox virus.
 39. The pharmaceutical composition of claim38, further comprising an adjuvant.